pubs.acs.org/jmc Article Synthesis, In Vitro Profiling, and In Vivo Evaluation of Benzohomoadamantane-Based Ureas for Visceral Pain: A New Indication for Soluble Epoxide Hydrolase Inhibitors Sandra Codony, José M. Entrena, Carla Calvó-Tusell, Beatrice Jora, Rafael González-Cano, Sílvia Osuna, Rubén Corpas, Christophe Morisseau, Belén Pérez, Marta Barniol-Xicota, Christian Griñán-Ferré, Concepción Pérez, María Isabel Rodríguez-Franco, Antón L. Martínez, M. Isabel Loza, Mercè Pallàs, Steven H. L. Verhelst, Coral Sanfeliu, Ferran Feixas, Bruce D. Hammock, José Brea, Enrique J. Cobos, and Santiago Vázquez* Cite This: J. Med. Chem. 2022, 65, 13660−13680 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: The soluble epoxide hydrolase (sEH) has been suggested as a pharmacological target for the treatment of several diseases, including pain-related disorders. Herein, we report further medicinal chemistry around new benzohomoadamantane-based sEH inhibitors (sEHI) in order to improve the drug metabolism and pharmacokinetics properties of a previous hit. After an extensive in vitro screening cascade, molecular modeling, and in vivo pharmacokinetics studies, two candidates were evaluated in vivo in a murine model of capsaicin-induced allodynia. The two compounds showed an anti-allodynic effect in a dose-dependent manner. Moreover, the most potent compound presented robust analgesic efficacy in the cyclophosphamide-induced murine model of cystitis, a well-established model of visceral pain. Overall, these results suggest painful bladder syndrome as a new possible indication for sEHI, opening a new range of applications for them in the visceral pain field. 1. INTRODUCTION AA to epoxyeicosatrienoic acids (EETs).7 EETs exhibit anti- Arachidonic acid (AA) is an essential ω-6 20 carbon hypertensive, anti-inflammatory, and anti-nociceptive proper- polyunsaturated fatty acid that is abundant in the phospholi- ties,8 but they are rapidly degraded by the soluble epoxide pids of cellular membrane. In response to a stimulus, hydrolase (sEH, EPHX2, E.C. 3.3.2.10) to the less active or inactive dihydroxyeicosatrienoic acids (DHETs). phospholipase A2 promotes its cleavage from the membrane Therefore, sEH inhibition may lead to elevated levels of and release into the cytosol, where it can be metabolized, EETs thereby maintaining their beneficial properties.9,10 leading to different classes of eicosanoids via three pathways Indeed, the use of selective sEH inhibitors (sEHI) in vivo (Figure 1).1,2 The cyclooxygenase (COX) pathway catalyzes models resulted in an increase of EETs levels and the reduction the production of prostaglandins, prostacyclins, and thrombox- anes, endowed with inflammatory properties. The lipoxygenase (LOX) pathway generates leukotrienes, which play a significant Received: April 1, 2022 part in the onset of asthma, arthritis, allergy, and inflamma- Published: October 12, 2022 tion.3 Both pathways have been extensively studied and targeted pharmaceutically.4−6 More recently, increasing attention is being paid to the third branch of the AA cascade, the cytochrome P450 (CYP) pathway that notably converts © 2022 The Authors. Published by American Chemical Society https://doi.org/10.1021/acs.jmedchem.2c00515 13660 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Figure 1. Simplified AA cascade. of blood pressure and inflammatory and pain states. Thus, sEH mouse microsomes, compound 6 was endowed with favorable has been suggested as a pharmacological target for the drug metabolism and pharmacokinetics (DMPK) properties treatment of several diseases, including pain-related disor- and showed efficacy in an in vivo murine model of acute ders.11−16 pancreatitis.21 Given that sEH presents a hydrophobic pocket, several Later on, in an effort for improving the DMPK properties of potent sEHI developed in the last years feature an adamantane piperidine 5, we designed a series of analogues where the urea moiety or an aromatic ring in their structure, such as AR9281, core was replaced by an amide group. Although most of these 1, and EC5026, 3, two of the sEHI that have reached clinical amides retained or even improved the inhibitory activity of trials.17,18 The first to enter was the adamantane-based their urea counterparts at the human and mouse enzymes (e.g., AR9281, by Arête Therapeutics, for the treatment of compound 7, Figure 2), only moderate improvements in hypertension in diabetic patients. However, it failed largely microsomal stabilities were found.22 because of its poor pharmacokinetic properties but also poor Herein, we report further medicinal chemistry around target residence time on sEH and only moderate potency on inhibitor 5. New piperidine derivatives retaining the urea the target.17 Very recently, EicOsis has replaced the group as the main pharmacophore, different substituents in the adamantane moiety of AR9281 by an aromatic ring for its C-9 position of the polycyclic scaffold (R in I), and a broad drug candidate EC5026, currently in phase 1 clinical trials for selection of substituents at the nitrogen atom of the piperidine the treatment of neuropathic pain.18 Interestingly, both clinical (R′ in I) were synthesized (Figure 2). After a screening candidates present similar structures: a left-hand side (lhs) cascade, two selected candidates with highly improved DMPK hydrophobic moiety (black), a urea group (green), a piperidine properties were subsequently studied in the murine model of residue (blue), and a right-hand side (rhs) acyl group (red). capsaicin-induced allodynia. Finally, the best compound was Also, EicOsis is currently advancing the analogue t-TUCB, 4, evaluated in a murine model of visceral pain. for veterinary clinical trials (Figure 2).19 Our recent observation that the lipophilic cavity of the 2. RESULTS AND DISCUSSION enzyme is flexible enough to accommodate polycyclic units 2.1. Design and Synthesis of New sEHI. For the larger than adamantane,20 led to the discovery of a new family preparation of the new sEHI, amines 8a−8g, previously of benzohomoadamantane-based ureas, such as 5 and 6, described by our group, were used as starting materials (Figure endowed with low nanomolar or even subnanomolar potencies 3).23−26 (Figure 2).21 Further in vitro studies with these compounds The synthesis of the novel urea-based sEHI was straightfor- demonstrated that while compound 5 presented moderate ward and involved the reaction of the benzohomoadamantane experimental solubility and very poor stability in human and amines 8a−g with triphosgene to obtain the corresponding 13661 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Figure 2. Structures and IC50 values in the human sEH of AR9281, 1, t-AUCB, 2, EC5026, 3, t-TUCB, 4, 5, 6, and 7 and general structure, I, of the new derivatives reported on this work. derivatives replacing the acetyl group of 5 by other fragments selected from previous other series of known sEHI to improve the microsomal stability.28,29 Compounds 9−12 were synthe- sized maintaining the methyl group in the position R of the benzohomoadamantane scaffold I and replacing the acetyl group of 5 by the propionyl, tetrahydro-2H-pyran-4-carbonyl, Figure 3. Benzohomoadamantane amines 8a−g used in this work. isopropylsufonyl, and cyclopropanecarbonyl groups, respec- tively (Scheme 1). The inhibitory activity against the human and murine enzymes of the new ureas was evaluated, as well as isocyanates II, followed by the addition of the required their stabilities in human and mouse microsomes (Table 1). substituted aminopiperidine of general structure III to form Gratifyingly, regardless of the substituent of the piperidine the final ureas 9−25 (Scheme 1). ring, all the compounds showed potency in the low nanomolar All the new compounds were fully characterized through their spectroscopic data and elemental analyses or high- or even subnanomolar ranges in both the human and murine performance liquid chromatography (HPLC)/mass spectrom- enzymes (Table 1). Indeed, the most potent compound, 12, etry (MS) (see the Experimental Section and the Supporting presented inhibitory activities in the subnanomolar range for Information for further details). both enzymes. However, except for 12, the microsomal 2.2. sEH Inhibition and Microsomal Stability. Com- stability of these new ureas was very poor and not improved pound 5 presented high inhibitory activities against the human from that of 5 (Table 1). and murine enzymes and moderate experimental aqueous Consequently, we moved to another strategy for improving solubility (38 μM), but unacceptable stability in human and the microsomal stability of the compounds, by exploring the C- murine microsomes (Table 1).21 Because the acyl chain of 9 position of the benzohomoadamantane scaffold, replacing piperidine-based sEHI is known to be a suitable position for the methyl group in 5 and 9−12 by other substituents, such as metabolism,27 we decided to explore first new piperidine halogen atoms or polar groups. The potency of these 13662 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Scheme 1. Synthesis of the New sEHIa a Reagents and conditions: (a) triphosgene, NaHCO3, DCM, 30 min; (b) DCM, overnight; and (c) n-BuLi, anhyd THF, anhyd DCM, overnight. See the Experimental Section and Supporting Information for further details. Table 1. IC50 in Human and Murine sEH, and Microsomal that the catalytic center of sEH is highly hydrophobic, the Stability Values of 5 and the New sEHI 9−12 compounds bearing a polar group in C-9, 23, and 24, presented higher IC50 values than 5. Of note, the most important drop in the inhibitory activity was produced by the replacement of the methyl group of 5 by the polar hydroxyl group, compound 23. On the other hand, when the methyl group was replaced by chlorine or fluorine atoms, the inhibitory activities against the human and murine enzymes were maintained or even improved, as most of them presented IC50 values in the low nanomolar or the subnanomolar range (Table 2). Next, the microsomal stability of the most potent compounds was evaluated. Pleasingly, all the compounds featuring halogen atoms in the R position of the benzohomoadamantane scaffold presented better stabilities in human and mice microsomes than their methyl counterparts (Table 2). Especially, the chlorinated compounds 16, 18, and 19 exhibited excellent microsomal stabilities in the two species. 2.3. In Silico Study: Molecular Basis of Benzohomoa- damantane/Piperidine-Based Ureas as sEH Inhibitors. Next, the mechanism of binding of two compounds with high inhibitory activity, that is, 15 (R = Cl, R′ = tetrahydro-2H- pyran-4-carbonyl) and 21 (R = F, R′ = tetrahydro-2H-pyran-4- carbonyl), was investigated with molecular dynamics (MD) simulations. sEHs present a flexible L-shaped active site pocket divided into three regions: the lhs and the rhs pockets that are connected by a central narrow channel defined by catalytic a Reported IC50 values are the average of three replicates. The residues Asp335, Tyr383, and Tyr466 (see Figure 4). Recently, fluorescent assay as performed here has a standard error between 10 we showed that bulky benzohomoadamantane groups occupy and 20%, suggesting that differences of twofold or greater are the lhs in urea-based sEHIs that present both adamantyl and significant. Because of limitations of the assay, it is difficult to phenyl moieties, for example, compound 6.21 However, distinguish among potencies <0.5 nM.30 bPercentage of remaining available X-ray structures of sEH in complex with piperidine- compound after 60 min of incubation with pooled human and mouse based ureas show that the piperidine group can also occupy the microsomes in the presence of NADPH at 37 °C. lhs.31 To determine the preferred binding mode of 15 and 21 that present both benzohomoadamantane and piperidine compounds was measured against the human and murine groups, we performed conventional MD simulations starting enzymes (Table 2). On the one hand, as expected considering from two possible orientations in the sEH active site predicted 13663 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Table 2. IC50 in Human and Murine sEH and Microsomal Stability Values of 13−25 a Reported IC50 values are the average of three replicates. The fluorescent assay as performed here has a standard error between 10 and 20%, suggesting that differences of twofold or greater are significant. Because of limitations of the assay, it is difficult to distinguish among potencies <0.5 nM.30 bPercentage of remaining compound after 60 min of incubation with pooled human and mouse microsomes in the presence of NADPH at 37 °C. cND: not determined. by molecular docking calculation (see the Experimental compounds present similar absolute binding affinities (−68.0 Section): (a) with the benzohomoadamantane in the lhs and and −69.4 kcal/mol for 15 and 21, respectively), which is in piperidine in the rhs (see Figure 4a, similar to adamantyl line with the similar IC50 values. To corroborate these results, based-urea in PDB 5AM3) and (b) the piperidine group is accelerated MD (aMD) simulations were performed to placed in lhs while benzohomoadamantane occupies rhs completely reconstruct the binding pathway of compound 15 (similar to piperidine based-urea in PDB 5ALZ).31 From into the sEH active site pocket (see Movie S1, Figure S1, and these MD simulations, the binding affinity of 15 and 21 was Experimental Section). This strategy is frequently used to estimated with molecular mechanics with generalized Born and predict substrate and inhibitor binding pathways in surface area solvation (MM/GBSA) calculations showing that enzymes.32,33 Spontaneous binding aMD simulations show the orientation shown in Figure 4a is −5.7 and −10.2 kcal/mol how the inhibitor is recognized in the lhs pocket by the more stable than the opposite orientation for compounds 15 benzohomodamantane scaffold and then extends through the and 21, respectively (see Table S2). When the benzohomoa- sEH binding site accommodating the benzohomoadamantane damantane occupies the lhs and the piperidine the rhs, both moiety in the lhs, while the piperidine counterpart lays in the 13664 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Figure 4. (a) Representative structure of compound 21 bound in the active site of sEH obtained from the most visited conformations along MD simulations. PDB ID 5AM3 has been used as the starting point for MD simulations. The benzohomoadamantane moiety occupies the lhs pocket while the piperidine group is placed in the rhs pocket. The central urea unit establishes hydrogen bonds with Asp335, Tyr466, and Tyr383. (b) Most relevant molecular interactions in the rhs. Average distances (in Å) obtained from three replicas of 500 ns of MD simulations are represented. Hydrogen bonds between the oxygens of the tetrahydropyran group of 21 and the hydrogen of the OH group of Ser415 is shown. The hydrophobic interaction average distances are computed between the terminal heavy atom of amino acid side chains and the centroid of each ring. Hydrogen bond distances between the carboxylic group of the catalytic Asp335 and the amide groups of the inhibitor and the distance between the carbonyl group of the urea inhibitor and the OH group of Tyr383 and Tyr466 residues. (c) Most relevant molecular interactions in the lhs. Average distances (in Å) obtained from the three replicas of 500 ns of MD simulations are represented. The CH−π interaction is calculated between the hydrogens of the benzohomoadamantane unit and the centroid of the benzoid ring of Trp336. The NH−π interaction is monitored between the amide hydrogen of Gln384 and the center of the aromatic ring of the benzohomoadamantane scaffold. rhs pocket. Considering these results, we conclude that the compound 15). MD simulations show that the inhibitor is orientation shown in Figure 4a is the preferred binding mode retained in the active site through three strong hydrogen bond of compounds 15 and 21. interactions between the urea moiety and the central channel To understand in more detail the molecular basis of the residues Asp335, Tyr383, and Tyr466 (see Figures 4b and S3). inhibitory mechanism of benzohomoadamantane/piperidine- In the rhs pocket, the piperidine group is stabilized through based ureas 15 and 21, the non-covalent interactions between persistent hydrophobic interactions with His494 and Met419, the selected inhibitors and the active site residues of sEH were while the tetrahydro-2H-pyran moiety is retained by the side studied (see Figure 4 for compound 21 and Figure S2 for chains of Leu417 and Trp525. The oxygen of tetrahydro-2H- 13665 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Table 3. Solubility and Permeability (PAMPA−BBB) Values, Cytotoxicity, and Inhibition of Pooled Human Cytochromes P450 Enzymes of Selected sEHI cytotoxicity LD50 (μM) cytochrome inhibitiond,e compound solubility (μM)a PAMPA−BBB PIb MTTc CYP 2C9 CYP 2C19 CYP 3A4 (7-BFC)f 14 18 CNS+ >100 >100 30 ± 4 46 ± 3 44 ± 1 15 57 CNS+ >100 >100 34 ± 1 38 ± 4 1±1 16 19 CNS+/− >100 >100 38 ± 1 1.48 μM 18 ± 1 17 19 CNS+/− >100 >100 34 ± 2 1.54 μM 1±1 18 16 CNS+/− >100 >100 54 ± 1 0.63 μM 2±1 19 17 CNS+/− >100 >100 43 ± 3 0.78 μM 3±2 21 95 CNS+/− >100 >100 30 ± 3 32 ± 4 2±2 22 92 CNS+ >100 >100 17 ± 2 26 ± 5 1±1 25 62 CNS+ >100 >100 12 ± 2 40 ± 1 0.70 μM a Solubility measured in a 1% DMSO: 99% PBS buffer solution. bCytotoxicity tested by PI staining after 24 h incubation in SH-SY5Y cells. c Cytotoxicity tested by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay after 24 h incubation in SH-SY5Y cells. dThe percent of cytochrome inhibition was tested at 10 μM. IC50 was calculated for those compounds that presented >50% of inhibition at 10 μM. eAt 10 μM, all the compounds inhibited <50% the cytochromes CYP1A2, CYP2D6, and CYP3A4 (DBF). fFor the study of CYP3A4, two different substrates were used benzyloxytrifluoromethylcoumarin (BFC) and dibenzylfluorescein (DBF). See the Experimental Section for further details. pyran ring establishes transient hydrogen bonds with Ser415 evaluation. Solubility, permeability through the blood−brain and is relatively solvent exposed (see Figure 4b). In the lhs barrier (BBB), cytotoxicity, and cytochrome inhibition of the pocket, the orientation of the benzohomoadmantane moiety is selected compounds 14−19, 21, 22, and 25 were exper- directed by the NH···π interaction between the Gln384 and imentally measured. In addition, we evaluated all the the aromatic ring of the polycyclic scaffold, which is synthesized compounds as pan assay interference compounds maintained along the MD simulations. Additionally, hydro- (PAINS) using SwissADME and FAFDrugs4 web tools.34,35 phobic interactions are established with the side chains of None of them gave positive as PAINS. Met339 and Trp336. This extensive network of hydrophobic While compounds 14, 16, 17, 18, and 19 exhibited limited interactions and hydrogen bonds in the sEH pocket is key to solubility, with values lower than 20 μM, compounds 15, 21, recognize and bind the inhibitor in the active site. 22, and 25 displayed good to excellent solubility values. Introducing a polar hydroxy group in the polycyclic scaffold Additionally, the selected compounds were further tested for (compound 23) significantly decreases the resulting inhibitory predicted brain permeation in the widely used in vitro parallel activity (see Table 2). To determine the molecular basis of this artificial membrane permeability assay−BBB (PAMPA−BBB) drop in activity, the binding modes of compounds 13 (R = Cl model.36 Compounds 14, 15, 22, and 25 showed CNS+ and R′ = acetyl and IC50 = 1.6 nM) and 23 (R = OH and R′ = proving their potential capacity to reach CNS, whereas the acetyl and IC50 = 207 nM) were compared with MD other compounds presented uncertain BBB permeation (CNS simulations. The incorporation of OH in the polycyclic +/−). Next, the cytotoxicity of the new sEHI was tested using scaffold causes a series of rearrangements in the lhs pocket the propidium iodide (PI) and MTT assays in SH-SY5Y cells. that destabilize the inhibitor bounds with the enzyme in the Interestingly, none of the selected compounds appeared active site (see Figure S4). In particular, the Thr360 side chain cytotoxic at the highest concentration tested (100 μM) establishes a hydrogen bond with the oxygen of the hydroxyl (Table 3). substituent of compound 23 that induces the rotation of the Finally, inhibition of several cytochrome P450 enzymes were benzohomoadamantane scaffold in the lhs pocket. This breaks measured, giving special attention to CYPs 2C19 and 2C9, as the NH−π interaction between Gln384 and the aromatic ring these isoforms are two of the main producers of EETs, the of 23 providing more flexibility to the benzohomadamantane substrates of the sEH.8 Unfortunately, compounds 16, 17, 18, moiety as compared to 13, 15, and 21, which may be related to and 19 inhibited significantly CYP 2C19. In contrast, the decreased activity (see Figure S5). In addition, the compounds 14, 15, 21, 22, and 25 did not significantly inhibit enhanced dynamism of the polycyclic scaffold allows the these subfamilies of cytochromes (Table 3). Additionally, transient entrance of few water molecules into the lhs pocket CYPs 2D6, 1A2, and 3A4 were also evaluated (Table S3). With (average number of water molecules 0.97 ± 0.96 for 23 and the only exception of 25, which inhibited CYP3A4 in the 0.31 ± 0.5 for 21, see Figure S6). Compound 24 (R = OCH3 submicromolar range, all the compounds showed IC50 values and R′ = acetyl, IC50 = 48 nM) that also present reduced higher than 10 μM (Tables 3 and S3). activity shows a similar behavior as 23 (see Figures S5 and S6). After performing the above-mentioned screening cascade, Therefore, the above-mentioned results and those previously three compounds, 15, 21, and 22, emerged as the more reported with related compounds,21 reveal that the presence of promising candidates. These compounds exhibited excellent a small, lipophilic group at C-9 of the benzohomodamantane inhibitory activities against the human and murine enzymes, scaffold is key for the stability and activity of benzohomoa- improved metabolic stability, good solubility, and did not damantane-based sEHIs at the molecular level. significantly inhibit cytochromes. Notwithstanding, hERG 2.4. Further DMPK Profiling of the Selected Inhib- inhibition and Caco-2 assays were also performed in order to itors. The halogen-substituted sEHI compounds that ex- additionally characterize them. None of the compounds hibited outstanding inhibitory activities and had more than significantly inhibit hERG at 10 μM, and they displayed 50% of the parent compound unaltered after incubation with moderate permeability in Caco-2 cells. Finally, they were human and/or murine microsomes were selected for further tested for selectivity against hCOX-2 and hLOX-5, two 13666 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Table 4. Permeability Values (Caco-2) and Inhibition of the hERG Channel of hLOX-5 and of hCOX-2 of the Selected Compounds 15, 21, and 22 permeability (Caco-2) papp (nm/s) Cpd A→B B→A ERa hERG channel inhibition (% inhib. 10 μM) IC50 hLOX-5 (μM)b IC50 hCOX-2 (μM)c 15 55.6 ± 0.7 171.6 ± 0.5 3.1 ± 0.3 1±2 >100 >10 21 32.9 ± 1 301.7 ± 26.5 9.2 ± 0.5 2±1 >100 >10 22 26.9 ± 2 235.4 ± 14.5 8.8 ± 0.2 1±2 >100 >10 a The efflux ratio was calculated as ER = (Papp B → A)/(Papp A → B). See the Experimental Section for further details. bIC50 in human LOX-5 (hLOX-5). See the Experimental Section for further details. cIC50 in human COX-2 (hCOX-2) performed by Eurofins (catalogue reference 4186). Scheme 2. Synthesis of the Probe 28a a See the Experimental Section for details. Figure 5. Target engagement and off-target profile of 28 in HEK293T cell lysates. (a) Fluorescence scan showing probe 28 labeling pattern in lysates, purified EPHX2- and EPHX2-spiked lysates, revealing that EPHX2 is the only target visibly outcompeted by the parent compound 15 and hence, the only target with high occupancy (Coomassie-stained gel in Figure S11). (b) Western blot analysis of selected proteins further confirms EPHX2 target engagement by 28 and proves that neither EPHX1, MAPK38, nor VEGFR are targeted by this compound. Compound 28 used at 10 μM and compound 15 used at 100 μM. enzymes involved in the AA cascade. Gratifyingly, they did not Next, we tested whether probe 28 could covalently bind present significant inhibition of these enzymes (Table 4). endogenously expressed human sEH in a complex proteome. 2.5. sEH Engagement and Off-Target Profile. Com- Hence, photoaffinity labeling was followed by incorporation of pound 28 was designed as a chemical probe with the objective an azide-TAMRA-Biotin tag via copper(I) azide alkyne to disturb the parent compound structure as little as possible. cycloaddition (CuAAc). This tag allows both visualization Important in this design was the knowledge that the piperidine and isolation of the probe’s protein targets. A fluorescent band nitrogen atom can be substituted without loss of biological at 72 KDa was identified as sEH via immunoblotting (Figures 5, S7, S9 and S10). activity. Therefore, a butynyl diazirinyl propionic acid Once the probe engagement of EPHX2 was confirmed, we minimalistic linker was coupled, via a straightforward amide determined the minimal probe labeling concentration using coupling reaction, to the piperidine nitrogen of 27, in turn purified recombinant human EPHX2 (Figure S8). The obtained from 8d through urea formation and Boc-removal minimal probe concentration was found to be 100 nM, (Scheme 2). The probe 28 was found to be a potent inhibitor which was then used to get insights in the selectivity of the with IC50 of 0.5 and 0.4 nM, for the human and mouse probe 28 and compound 15. Although it was observed that enzymes, respectively. probe 28 labeled multiple bands, competition with the parent 13667 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Table 5. Pharmacokinetic Parameters in Male CD1 Mice for Compounds 15 and 21 After 5 mg/kg sc Administrationa compound Dose HL (h) Tmax (h) Cmax (μg/mL) AUClast (μg*h/mL) AUCINF (μg*h/mL) 15 5 mg/Kg 3.42 0.75 1.2 2.4 2.5 21 5 mg/Kg 0.70 0.25 19.1 13.5 13.6 a See the Experimental Section and Tables S4 and S5 and Figures S12 and S13 in the Supporting Information. Figure 6. Reduction of capsaicin-induced secondary mechanical hypersensitivity in mice by the systemic administration of AS2586114, and compounds 15 and 21, is due to sEH inhibition. The data shown represent the effect of the sc administration of AS2586114, 15, and 21 administered alone or associated with the CYP450 oxidase inhibitor MS-PPOH (sc) on paw withdrawal latency in mice-treated intra-plantarly (i.pl.) with capsaicin. Each bar and vertical line represent the mean ± SEM of the values obtained in 8−10 animals. Statistically significant differences: **p < 0.01 between nonsensitized mice (open bar) and the other experimental groups; #p < 0.05 and ##p < 0.01 between capsaicin- treated mice injected with the sEHI or their solvent (black bar); ++p < 0.01 sEHI-treated mice associated or not with MS-PPOH (one-way ANOVA followed by Student−Newman−Keuls test). compound 15 shows competition of only EPHX2, illustrating 2.7. In Vivo Efficacy Studies. A first in vivo efficacy study that this is the sole target with high occupancy and that the was performed in a capsaicin-induced secondary mechanical other bands are non-specific labeling events by the probe hypersensitivity (allodynia) model in mice. It is well known (Figure 5). To further confirm the selective character of 15, we that the increase in sensitivity to mechanical stimulation in the wanted to exclude p38 mitogen-activated protein kinase (p38 area surrounding capsaicin injection results from central MAPK) and pro-angiogenic kinase vascular endothelial growth sensitization,41 which is a key process in chronic pain factor receptor-2 (VEGFR2) as targets because some urea- development and maintenance.42 In our experimental con- based sEHI are reported to show cross-reactivity with these ditions, mice markedly decreased their paw withdrawal latency proteins.37−39 In addition, we also aimed to exclude membrane to mechanical stimulation after capsaicin administration bound microsomal epoxide hydrolase as a possible off-target.40 (Figure 6), denoting the development of mechanical allodynia. To this end, we performed pull-down experiments and The sc administration of the prototypic, brain-penetrant,43−46 immunoblotting with specific antibodies. These experiments sEHI AS2586114 induced a dose-dependent reversion of the confirmed that none of these proteins are targets of 28, capsaicin-induced mechanical hypersensitivity reaching a full underlining its selectivity (Figure 5b). reversal of sensory hypersensitivity at 10 mg/kg (Figure 6). 2.6. Pharmacokinetic Study of Compounds 15 and The sc administration of compounds 15 and 21 fully inhibited 21. Overall, compounds 15 and 21, with similar DMPK mechanical hypersensitivity in a dose-dependent manner and properties and structures, were selected for in vivo studies. First, a study was conducted in order to determine the with a much higher potency than AS2586114, reaching full pharmacokinetic profile in the plasma of compounds 15 and reversal of sensory gain with 5 mg/kg for compound 15 and 21 when administered by a subcutaneous (sc) route at a single even with a dose as low as 1.25 mg/kg for compound 21 dose of 5 mg/kg. As shown in Table 5, absorption of 21 is fast, (Figure 6), in spite of its limited predicted BBB permeability reaching Cmax (19.1 μg/mL) at 15 min after dosing. The (as previously commented). Importantly, the administration of compound disappeared from the plasma progressively and half- N-methanesulfonyl-6-(2-proparyloxyphenyl)hexanamide (MS- life (HL) was calculated to be around 0.7 h. In the case of 15, PPOH), an inhibitor of microsomal CYP450s, which is Cmax (1.2 μg/mL) was 15 times lower than that of 21, however, responsible for the production of EETs,47 fully abolished the showing a higher HL (3.4 h). For both compounds, the narrow effect of not only AS2586114 but also those induced by differences in AUC0t and AUC0∞ showed complete exposure compounds 15 and 21 (Figure 6). These results strongly and good bioavailability. Although 21 demonstrated better suggest that the three tested compounds induced the reversal bioavailability characteristics than 15 both compounds were of capsaicin-induced mechanical hypersensitivity through the subsequently evaluated in vivo efficacy studies. in vivo inhibition of sEH. 13668 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Given that the tested compounds induced ameliorative effects on this behavioral model of central sensitization attributable to sEH inhibition, we tested the effect of compound 21 (the most potent compound among the sEHI evaluated), in a model of pathological pain. Specifically, cyclophosphamide (CTX)-induced cystitis because it has been used as a model of interstitial cystitis/bladder pain syndrome,48 and it is known that pain induced by this disease has a strong component of central sensitization in both humans and rodents.49,50 In our experimental conditions, mice treated with CTX showed a significant increase in the pain behavioral score in comparison to mice treated with the vehicle (Figure 7a). The sc administration of compound 21 (0.63−2.5 mg/kg) significantly reduced this pain-related score in a dose- dependent manner (Figure 7a). In addition, animals administered with the CTX vehicle showed a marked reduction in their mechanical threshold in the abdomen, denoting the development of referred hyperalgesia (Figure 7b). The sc treatment with compound 21 also reversed, in a dose- dependent manner, the mechanical referred hyperalgesia induced by CTX (Figure 7b). The administration of MS- PPOH fully reversed the effect of compound 21 in either the pain-related behaviors as in referred hyperalgesia (Figure 7a,b, respectively), mirroring the results obtained on capsaicin- induced secondary hyperalgesia and suggesting that compound 21 exerted its in vivo effects on pain through sEH inhibition. To our knowledge, there are no previous studies exploring the role of sEHI on visceral pain. Therefore, our results suggest interstitial cystitis/pain bladder syndrome as a possible new indication for inhibitors of sEH. 3. CONCLUSIONS sEH is a suitable target for several inflammatory and pain- related diseases. In this work, we report further medicinal chemistry around new benzohomoadamantane-based piper- idine derivatives, analogues of the clinical candidates AR9281 Figure 7. Effects of compound 21 on pain-related behaviors and and EC5026. The introduction of a halogen atom in the referred mechanical hyperalgesia induced by CTX. (a) Behavioral position C-9 of the benzohomoadamantane scaffold led to very score was recorded at 30 min intervals over the 150−240 min potent compounds with improved DMPK properties. The in observation period after the intraperitoneal (ip) injection of (CTX, vitro profiling of these new sEHI (solubility, cytotoxicity, 300 mg/kg) or its vehicle. (b) 50% mechanical threshold was metabolic stability, CYP450s, hLOX-5, hCOX-2, and hERG evaluated by stimulation of the abdomen with von Frey filaments at 240 min after the administration CTX or its vehicle and was used as inhibition) allowed one to select two suitable candidates for in an index of referred hyperalgesia. Each bar and vertical line represents vivo efficacy studies. The administration of compounds 15 and the mean ± SEM of values obtained in at least six animals per group. 21 reduced pain in the capsaicin-induced murine model of Statistically significant differences: **p < 0.01, between nonsensitized allodynia in a dose-dependent manner and outperformed mice (open bar) and the other experimental groups; #p < 0.05, ##p < AS2586114. Moreover, compound 21 was tested in a CTX- 0.01 between CTX-treated mice injected with the sEHI or their induced murine model of cystitis, revealing its robust analgesic solvent (black bar); ++p < 0.01 mice injected with compound 21 effect. Hence, this study opens a whole range of applications of associated or not with MS-PPOH (one-way ANOVA followed by the benzohomoadamantane-based sEHIs in pain and likely Student−Newman−Keuls test). other fields. showed a sharp melting point and a single spot on TLC. Purity >95% 4. EXPERIMENTAL SECTION of all final compounds was assessed by the integration of LC 4.1. Chemical Synthesis. Commercially available reagents and chromatograms. Melting points were determined in open capillary solvents were used without further purification unless stated tubes with a MFB 595010M Gallenkamp. 400 MHz 1H and 100.6 otherwise. Preparative normal phase chromatography was performed MHz 13C NMR spectra were recorded on a Varian Mercury 400 or on on a CombiFlash Rf 150 (Teledyne Isco) with pre-packed RediSep Rf a Bruker 400 Avance III spectrometers. 500 MHz 1H NMR spectra silica gel cartridges. Thin-layer chromatography was performed with were recorded on a Varian Inova 500 spectrometer. The chemical aluminum-backed sheets with silica gel 60 F254 (Merck, ref 1.05554), shifts are reported in ppm (δ scale) relative to internal and spots were visualized with UV light and 1% aqueous solution of tetramethylsilane, and coupling constants are reported in Hertz KMnO4. HPLC purification was performed on a Prominence ultra- (Hz). Assignments given for the NMR spectra of selected new fast liquid chromatography system (Shimadzu) using a Waters X- compounds have been carried out on the basis of DEPT, COSY 1H/ bridge 150 mm C18 prep column with a gradient of acetonitrile in 1H (standard procedures), and COSY 1H/13C (gHSQC and gHMBC water (with 0.1% trifluoroacetic acid) over 32 min. All compounds sequences) experiments. IR spectra were run on PerkinElmer 13669 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article spectrum RX I, PerkinElmer spectrum TWO, or Nicolet Avatar 320 aminopiperidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone (215 mg, FT-IR spectrophotometers. Absorption values are expressed as 1.01 mmol). The reaction mixture was stirred at room temperature wavenumbers (cm−1); only significant absorption bands are given. overnight, and the solvent was evaporated under vacuum to obtain a High-resolution mass spectrometry (HRMS) analyses were performed yellow residue (534 mg). Column chromatography (SiO2, DCM/ with an LC/MSD TOF Agilent Technologies spectrometer. The methanol mixtures) gave urea 10 (207 mg, 45% yield) as a white elemental analyses were carried out in a Flash 1112 series Thermo solid, mp 224−225 °C. IR (NaCl disk): 3357, 3064, 3017, 2945, Finnigan elemental microanalyzer (A5) to determine C, H, N, and S. 2919, 2850, 1640, 1614, 1553, 1493, 1446, 1361, 1344, 1320, 1278, The structure of all new compounds was confirmed by elemental 1261, 1238, 1211, 1126, 1089, 1068, 1018, 984, 941, 874, 818, 759, analysis and/or accurate mass measurement, IR, 1H NMR, and 13C 733 cm−1. 1H NMR (400 MHz, CDCl3): δ 0.90 (s, 3H, C9−CH3), NMR. The analytical samples of all the new compounds, which were 1.17 [m, 2H, 3′(5′)-Hax], 1.50−1.65 [complex signal, 6H, 3″(5″)-Hax, subjected to pharmacological evaluation, possessed purity ≥95% as 10(13)-H2], 1.79 (s, 2H, 8-H), 1.82−1.90 [complex signal, 3H, 5′-Heq evidenced by their elemental analyses (Table S1) or HPLC/UV. or 3′-Heq, 3″(5″)-Heq], 1.94 [d, J = 12.8 Hz, 2H, 6(12)-Hax], 2.03− HPLC/UV were determined with a HPLC Agilent 1260 Infinity II 2.16 [complex signal, 3H, 6(12)-Heq, 3′-Heq or 5′-Heq], 2.65−2.79 LC/MSD coupled to a photodiode array. 5 μL of sample 0.5 mg/mL (complex signal, 2H, 2′-Hax or 6′-Hax, 4″-H), 3.00−3.17 [complex in methanol/acetonitrile were injected, using an Agilent Poroshell 120 signal, 3H, 6′-Hax or 2′-Hax, 5(11)-H], 3.43 [m, 2H, 2″(6″)-Hax], EC-C18, 2.7 μm, 50 mm × 4.6 mm column at 40 °C. The mobile 3.69−3.88 (complex signal, 2H, 4′-H, 2′-Heq or 6′-Heq), 3.99 [m, 2H, phase was a mixture of A = water with 0.05% formic acid and B = 2″(6″)-Heq], 4.48 (m, 2H, 2′-Heq or 6′-Heq), 7.02 [m, 2H, 1(4)-H], acetonitrile with 0.05% formic acid, with the method described as 7.06 [m, 2H, 2(3)-H]. 13C NMR (100.6 MHz, CDCl3): δ 29.1 [CH2, follows: flow 0.6 mL/min, 5% B−95% A 3 min, 100% B 4 min, and C3″(5″)], 32.3 (CH3, C9−CH3), 32.4 (CH2, C5′ or C3′), 33.7 (C, 95% B−5% A 1 min. Purity is given as % of absorbance at 220 nm. C9), 34.1 (CH2, C3′ or C5′), 37.6 (CH, C4″), 39.9 [CH2, C6(12)], 4.1.1. 1-(9-Methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- 41.1 [CH, C5(11)], 41.2 [CH2, C10(13)], 44.3 [CH2, C2′(6′)], 47.0 dimethanobenzo[9]annulen-7-yl)-3-(1-propionylpiperidin-4-yl)- (CH, C4′), 48.0 (CH2, C8), 53.5 (C, C7), 67.1 [CH2, C2″(6″)], urea (9). To a solution of 9-methyl-5,6,8,9,10,11-hexahydro-7H- 126.2 [CH, C2(3)], 127.9 [CH, C1(4)], 146.3 [C, C4a(11a)], 156.3 5,9:7,11-dimethanobenzo[9]annulen-7-amine hydrochloride (464 mg, (C, NHCONH), 172.8 (C, NCOR). Anal. Calcd for C28H39N3O3: C, 1.76 mmol) in DCM (10 mL), saturated aqueous NaHCO3 solution 72.23; H, 8.44; N, 9.02. Found: C, 72.33; H, 8.40; N, 8.83. HRMS (10 mL) and triphosgene (193 mg, 0.65 mmol) were added. The calcd for [C28H39N3O3 + H]+, 466.3064; found, 466.3065. biphasic mixture was stirred at room temperature for 30 min, then the 4.1.3. 1-[1-(Isopropylsulfonyl)piperidin-4-yl]-3-(9-methyl- two phases were separated, and the organic layer was washed with 5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen- brine (5 mL), dried over anhyd Na2SO4, filtered, and evaporated 7-yl)urea (11). To a solution of 9-methyl-5,6,8,9,10,11-hexahydro- under vacuum to obtain 1−2 mL of a solution of the isocyanate in 7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine hydrochloride (300 DCM. To this solution was added 1-(4-aminopiperidin-1-yl)propan- mg, 1.14 mmol) in DCM (6 mL) and saturated aqueous NaHCO3 1-one (350 mg, 2.24 mmol). The reaction mixture was stirred at room solution (4 mL) was added triphosgene (169 mg, 0.57 mmol). The temperature overnight and the solvent was evaporated under vacuum biphasic mixture was stirred at room temperature for 30 min, then the to obtain a white solid (741 mg). Column chromatography (SiO2, two phases were separated, and the organic layer was washed with DCM/methanol mixtures) gave urea 9 (597 mg, 83% yield) as a brine (5 mL), dried over anhyd Na2SO4, filtered, and evaporated white solid. The analytical sample was obtained by crystallization from under vacuum to obtain 1−2 mL of a solution of the isocyanate in hot EtOAc and DCM (300 mg), mp 207−208 °C. IR (NaCl disk): DCM. 3357, 2917, 2859, 1644, 1620, 1556, 1493, 1450, 1361, 1344, 1319, To a solution of 1-(isopropylsulfonyl)piperidin-4-amine (233 mg, 1264, 1221, 1132, 1068, 1024, 971, 949, 758 cm−1. 1H NMR (400 1.13 mmol) in anhyd THF (5 mL) under an argon atmosphere at MHz, CDCl3): δ 0.90 (s, 3H, C9−CH3), 1.11 (t, J = 7.2 Hz, 3H, −78 °C was added dropwise a solution of n-butyllithium (2.5 M in COCH2CH3), 1.14 [m, 2H, COCH2CH3, 5′(3′)-Hax], 1.52 [d, J = hexanes, 0.59 mL, 1.47 mmol) during 20 min. After the addition, the 13.4 Hz, 2H, 10(13)-Hax], 1.62 [dd, J = 13.4 Hz, J′ = 6.0 Hz, 2H, mixture was tempered to 0 °C using an ice bath. This solution was 10(13)-Heq], 1.77−1.86 (complex signal, 3H, 8-H2, 5′-Heq or 3′-Heq), added carefully to the solution of the isocyanate from the previous 1.93 [d, J = 12.8 Hz, 2H, 6(12)-Hax], 2.02 (d, J = 12.0 Hz, 1H, 3′-Heq step cooled to 0 °C, under an argon atmosphere. The reaction or 5′-Heq), 2.12 [dd, J = 12.8 Hz, J′ = 6.0 Hz, 2H, 6(12)-Heq], 2.32 mixture was stirred at room temperature overnight. Methanol (2 mL) (m, 2H, COCH2CH3), 2.70 (m, 1H, 2′-Hax or 6′-Hax), 3.00−3.12 was then added to quench any unreacted n-butyllithium. The solvents [complex signal, 3H, 5(11)-H, 6′-Hax or 2′-Hax], 3.70−3.77 (complex were evaporated under vacuum to give an orange gum (506 mg). This signal, 2H, 4′-H, 6′-Heq or 2′-Heq), 4.47 (d, J = 13.6 Hz, 1H, 2′-Heq or residue was dissolved in EtOAc (10 mL) and washed with 2 N HCl 6′-Heq), 4.64−4.72 (complex signal, 2H, C7−NH, C4′-NH), 7.02 [m, solution (2 × 5 mL) and the organic layer was dried over anhyd 2H, 1(4)-H], 7.05 [m, 2H, 2(3)-H]. 13C NMR (100.6 MHz, CDCl3): Na2SO4, filtered, and concentrated in vacuum to obtain a white gum δ 9.7 (CH3, COCH2CH3), 26.6 (CH2, COCH2CH3), 32.3 (CH3, (241 mg). Column chromatography (SiO 2, DCM/methanol C9−CH3), 32.4 (CH2, C3′ or C5′), 33.6 (C, C9), 33.9 (CH2, C5′ or mixtures) gave a white solid. Crystallization from hot DCM/pentane C3′), 39.9 [CH2, C6(12)], 40.9 (CH2, C6′ or C2′), 41.1 [CH, provided urea 11 (66 mg, 13% yield) as a white solid, mp 218−219 C5(11)], 41.2 [CH2, C10(13)], 44.5 (CH2, C2′ or C6′), 46.7 (CH, °C. IR (NaCl disk): 3364, 3062, 3013, 2946, 2920, 2854, 1710, 1638, C4′), 48.0 (CH2, C8), 53.4 (C, C7), 126.2 [CH, C2(3)], 127.9 [CH, 1553, 1494, 1453, 1361, 1320, 1305, 1266, 1249, 1232, 1168, 1134, C1(4)], 146.3 [C, C4a(11a)], 156.5 (C, NHCONH), 172.4 1091, 1045, 943, 881, 841, 759, 732, 666, 593, 555 cm−1. 1H NMR (NCOCH2CH3). Anal. Calcd for C25H35N3O2·0.25 H2O: C, 72.52; (400 MHz, CDCl3): δ 0.90 (s, 3H, C9−CH3), 1.31 [d, J = 6.8 Hz, H, 8.64; N, 10.15. Found: C, 72.65; H, 8.49; N, 9.82. HRMS calcd for 6H, CH(CH3)2], 1.36 [dq, J = 12.0 Hz, J′ = 4.0 Hz, 3′(5′)-Hax], 1.52 [C25H35N3O2 + H]+, 410.2802; found, 410.2801. [d, J = 13.2 Hz, 2H, 10(13)-Hax], 1.61 [m, 2H, 10(13)-Heq], 1.79 (s, 4.1.2. 1-(9-Methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- 2H, 8-H), 1.92−1.97 [complex signal, 4H, 3′(5′)-Heq, 6(12)-Hax], dimethanobenzo[9]annulen-7-yl)-3-(1-(tetrahydro-2H-pyran-4- 2.12 [dd, J = 12.8 Hz, J′ = 6.4 Hz, 2H, 6(12)-Heq], 2.92 [m, 2H, carbonyl)piperidin-4-yl)urea (10). To a solution of 9-methyl- 2′(6′)-Hax], 3.04 [t, J = 6.4 Hz, 2H, 5(11)-H], 3.15 (sept, J = 6.8 Hz, 5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7- 1H, CH(CH3)2), 3.67 (m, 1H, 4′-H), 3.78 [dm, J = 13.2 Hz, 2H, amine hydrochloride (258 mg, 0.98 mmol) in DCM (4 mL), 2′(6′)-Heq], 4.35 (s, 1H, C7−NH), 4.41 (d, J = 8.0 Hz, C4′-NH), saturated aqueous NaHCO3 solution (4 mL) and triphosgene (107 7.03 [m, 2H, 1(4)-H], 7.06 [m, 2H, 2(3)-H]. 13C NMR (100.6 MHz, mg, 0.36 mmol) were added. The biphasic mixture was stirred at CDCl3): δ 16.7 [CH3, CH(CH3)2], 32.3 (CH3, C9−CH3), 33.4 room temperature for 30 min, then the two phases were separated, [CH2, C3′(5′)], 33.7 (C, C9), 39.8 [CH2, C6(12)], 41.1 [CH, and the organic layer was washed with brine (2 mL), dried over anhyd C5(11)], 41.2 [CH2, C10(13)], 45.7 [CH2, C2′(6′)], 46.6 (CH, Na2SO4, filtered, and evaporated under vacuum to obtain 1−2 mL of C4′), 48.0 (CH2, C8), 53.4 [CH, CH(CH3)2], 53.5 (C, C7), 126.2 a solution of the isocyanate in DCM. To this solution was added (4- [CH, C2(3)], 127.9 [CH, C1(4)], 146.3 [C, C4a(11a)], 156.2 (C, 13670 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article NHCONH). Anal. Calcd for C25H37N3O3S: C, 65.33; H, 8.11; N, (CH2, C12 or C6), 40.7 (CH2, C2′ or C6′), 41.2 [CH, C5(11)], 9.14. Found: C, 65.41; H, 8.31; N, 8.93. HRMS calcd for 44.47 (CH2, C10 or C13), 44.50 (CH2, C13 or C10), 45.4 (CH2, C6′ [C25H37N3O3S + H]+, 460.2628; found, 460.2623. or C2′), 46.6 (CH, C4′), 50.8 (CH2, C8), 55.5 (C, C7), 69.5 (C, 4.1.4. 1-(9-Methyl-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- C9), 126.8 [CH, C2(3)], 128.1 [CH, C1(4)], 144.7 (CH, C4a or dimethanobenzo[9]annulen-7-yl)-3-(1-(cyclopropanecarbonyl)- C11a), 144.8 (CH, C11a or C4a), 156.2 (C, NHCONH), 169.17 (C, piperidin-4-yl)urea (12). To a solution of 9-methyl-5,6,8,9,10,11- COCH3). Anal. Calcd for C23H30ClN3O2·0.75 ethyl acetate: C, 64.78; hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine hydro- H, 7.53; N, 8.72. Found: C, 64.73; H, 7.56; N, 8.89. HRMS calcd for chloride (112.5 mg, 0.43 mmol) in DCM (6 mL), saturated aqueous [C23H30ClN3O2 + H]+, 416.2099; found, 416.2100. NaHCO3 solution (5 mL) and triphosgene (93.8 mg, 0.16 mmol) 4.1.6. 1-(9-Chloro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- were added. The biphasic mixture was stirred at room temperature for dimethanobenzo[9]annulen-7-yl)-3-(1-propionylpiperidin-4-yl)- 30 min and then the two phases were separated and the organic layer urea (14). To a solution of 9-chloro-5,6,8,9,10,11-hexahydro-7H- was washed with brine (5 mL), dried over anhyd Na2SO4, filtered, and 5,9:7,11-dimethanobenzo[9]annulen-7-amine hydrochloride (150 mg, evaporated under vacuum to obtain 2−3 mL of a solution of the 0.53 mmol) in DCM (4 mL) and saturated aqueous NaHCO3 isocyanate in DCM. To this solution was added (4-aminopiperidin-1- solution (3 mL), triphosgene (56 mg, 0.19 mmol) was added. The yl)(tetrahydro-2H-pyran-4-yl)methanone (72 mg, 0.43 mmol). The biphasic mixture was stirred at room temperature for 30 min, then the mixture was stirred overnight at room temperature, and the solvent two phases were separated, and the organic one was washed with was then evaporated. Column chromatography (SiO2, DCM/ brine (3 mL), dried over anhyd Na2SO4, filtered, and evaporated methanol mixtures) provided urea 12 as a white solid (60 mg, 33% under vacuum to obtain 1−2 mL of a solution of isocyanate in DCM. yield), mp 115−120 °C. IR (ATR): 3341, 2899, 1633, 1607, 1549, To this solution was added 1-(4-aminopiperidin-1-yl)propan-1-one 1448, 1311, 1222, 1128, 1064, 1027, 979, 756. 1H NMR (400 MHz, (83 mg, 0.53 mmol). The mixture was stirred overnight at room CDCl3): δ 0.72 [dd, 2H, J = 6.0 Hz, J′ = 2.0 Hz, 8′(9′)-Hax], 0.90 (s, temperature, and the solvent was then evaporated. Column 3H, 9′-H), 0.93 [m, 2H, 8′(9′)-Heq], 1.20 [m, 2H, 3′(5′)-Heq], 1.52 chromatography (SiO2, DCM/methanol mixtures) provided an [d, 2H, J = 13.2 Hz, 10(13)-Hax], 1.63 [dd, 2H, J = 13.2 Hz, J′ = 5.6 orange solid. The analytical sample was obtained by a crystallization Hz, 10(13)-Heq], 1.73 (m, 2H, 7′-H), 1.80 [m, 3H, 8-H, 3′ or 5′-Heq], from hot ethyl acetate/pentane mixtures to obtain a urea 14 as a 1.95 [d, 2H, J = 12.8 Hz, 6(12)-Hax], 2.04 [d, 1H, J = 12.0 Hz, 3′ or yellowish solid (79 mg, 35% yield), mp 155−156 °C. IR (ATR): 5′-Heq], 2.13 [dd, 2H, J = 12.0 Hz, J′ = 6.4 Hz, 6(12)-Heq], 2.73 [t, 3359, 2924, 2852, 1681, 1652, 1637, 1612, 1565, 1447, 1373, 1356, 1H, J = 11.6 Hz, 2′ or 6′-Hax], 3.04 [t, 2H, J = 12.0 Hz, 5(11)-H], 1322, 1297, 1263, 1221, 1134, 1075, 1045, 1022, 967, 946, 908, 804, 3.20 [t, 1H, J = 12 Hz, 2′ or 6′-Hax], 3.75 (m, 1H, 4′-H), 4.1 [d, 1H, J 755, 618, 559 cm−1. 1H NMR (400 MHz, CDCl3): δ 1.11 (t, J = 7.2 = 10.8 Hz, 2′ or 6′-Heq], 4.4 [d, 1H, J = 10 Hz, 2′ or 6′-Heq], 4.5 [d, Hz, 3H, COCH2CH3), 1.13 [m, 2H, 5′(3′)-Hax], 1.84 (d, J = 12.8 Hz, 1H, J = 10.8 Hz, NHCONH], 4.6 (s, 1H, NHCONH), 7.03 [m, 2H, 1H, 5′-Heq or 3′-Heq), 1.94 [d, J = 12.8 Hz, 2H, 6(12)-Hax], 2.00 (d, J 1(4)-H], 7.05 [m, 2H, 2(3)-H]. 13C NMR (100.5 MHz, CDCl3): δ = 12.4 Hz, 3′-Heq or 5′-Heq), 2.14 [d, J = 13.2 Hz, 2H, 10(13)-Hax], 7.49 [CH2, C8′(9′)], 11.14 (CH, C7′), 32.43 (CH3, C9′), 32.53 2.21 [m, 2H, 6(12)-Heq], 2.29−2.40 [complex signal, 4H, 10(13)-Heq, (CH2, C3′ or 5′), 33.82 (CH2, C3′ or 5′), 40.03 [CH2, C6(12)], COCH2CH3], 2.48 (m, 2H, 8-H), 2.70 (m, 1H, 2′-Hax or 6′-Hax), 41.26 [CH, C5(11)], 41.35 [CH2, C10(13)], 41.66 [CH2, C2′ or 6′], 3.08 (m, 1H, 6′-Hax or 2′-Hax), 3.14 [t, J = 6.4 Hz, 2H, 5(11)-H], 44.71 [CH2, C2′or 6′], 47.04 (CH, C4′), 48.13 (CH2, C8), 53.58 (C, 3.68−3.82 (complex signal, 2H, 6′-Heq or 2′-Heq, 4′-H), 4.45 (dm, J = C7), 126.33 [CH, C(1)4], 128.07 [CH, C2(3)], 146.49 {C, 13.6 Hz, 1H, 2′-Heq or 6′-Heq), 4.68 (d, J = 8.0 Hz, 1H, C4′-NH), C4a(11a)], 156.57 (C, NHCONH), 172.22 (C, NCOR). Anal. 4.75 (s, 1H, C7−NH), 7.05 [m, 2H, 1(4)-H], 7.10 [m, 2H, 2(3)-H]. 13 Calcd for C26H35N3O2·0.1 CH2Cl2: C, 72.89; H, 8.25; N, 9.77. C NMR (100.6 MHz, CDCl3): δ 9.7 (CH3, COCH2CH3), 26.6 Found: C, 73.08; H, 8.23; N, 9.53. HRMS calcd for [C26H35N3O2 + (CH2, COCH2CH3), 32.4 (CH2, C5′ or C3′), 33.9 (CH2, C3′ or H]+, 422.2802; found, 422.2808. C5′), 38.9 [CH2, C6(12)], 40.9 (CH2, C2′ or C6′), 41.2 [CH, 4.1.5. 1-(1-Acetylpiperidin-4-yl)-3-(9-chloro-5,6,8,9,10,11-hexa- C5(11)], 44.5 [2 CH2, C10(13), C6′ or C2′], 46.7 (CH, C4′), 50.8 hydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea (13). To (CH2, C8), 55.5 (C, C7), 69.5 (C, C9), 126.8 [CH, C2(3)], 128.1 a solution of 9-chloro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- [CH, C1(4)], 144.8 [C4a(11a)], 156.1 (NHCONH), 172.5 dimethanobenzo[9]annulen-7-amine hydrochloride (150 mg, 0.53 (NCOR). HRMS calcd for [C24H32ClN3O2 + H]+, 430.2256; mmol) in DCM (3 mL), saturated aqueous NaHCO3 solution (3 mL) found, 430.2253. Anal. Calcd for C24H32ClN3O2·0.75 H2O: C, and triphosgene (58 mg, 0.20 mmol) were added. The biphasic 65.00; H, 7.61; N, 9.47. Found: C, 65.27; H, 7.51; N, 9.15. mixture was stirred at room temperature for 30 min, then the two 4.1.7. 1-(9-Chloro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- phases were separated, and the organic layer was washed with brine (3 dimethanobenzo[9]annulen-7-yl)-3-(1-(tetrahydro-2H-pyran-4- mL), dried over anhyd Na2SO4, filtered, and evaporated under carbonyl)piperidin-4-yl)urea (15). To a solution of 9-chloro- vacuum to obtain 1−2 mL of a solution of the isocyanate in DCM. To 5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7- this solution was added 1-(4-aminopiperidin-1-yl)ethan-1-one (90 amine hydrochloride (130 mg, 0.46 mmol) in DCM (4 mL) and mg, 0.63 mmol). The reaction mixture was stirred at room saturated aqueous NaHCO3 solution (3 mL), triphosgene (50 mg, temperature overnight, and the solvent was evaporated under vacuum 0.17 mmol) was added. The biphasic mixture was stirred at room to obtain a white solid (204 mg). Column chromatography (SiO2, temperature for 30 min, then the two phases were separated, and the DCM/methanol mixtures) gave urea 13 (115 mg, 52% yield) as a organic one was washed with brine (3 mL), dried over anhyd Na2SO4, white solid, mp 209−210 °C. IR (NaCl disk): 3358, 3019, 2926, filtered, and evaporated under vacuum to obtain 1−2 mL of a solution 2855, 1644, 1619, 1556, 1494, 1452, 1358, 1319, 1301, 1268, 1228, of isocyanate in DCM. To this solution was added (4-aminopiperidin- 1206, 1135, 1090, 1050, 991, 969, 947, 802, 761, 735 cm−1. 1H NMR 1-yl) (tetrahydro-2H-pyran-4-yl)methanone (97 mg, 0.46 mmol). (400 MHz, CDCl3): δ 1.15 (m, 1H, 5′-Hax or 3′-Hax), 1.18 [m, 1H, The mixture was stirred overnight at room temperature, and the 3′-Hax or 5′-Hax], 1.85 (d, J = 13.6 Hz, 1H, 5′-Heq or 3′-Heq), 1.93 [d, solvent was then evaporated. Column chromatography (SiO2, DCM/ J = 13.2 Hz, 2H, 6(12)-Hax], 2.02 (m, 1H, 3′-Heq or 5′-Heq), 2.06 (s, methanol mixtures) provided urea 15 as a yellowish solid (90 mg, 3H, COCH3), 2.15 [d, J = 13.6 Hz, 2H, 10(13)-Hax], 2.21 [m, 2H, 41% yield). The analytical sample was obtained by washing the 6(12)-Heq], 2.35 [dd, J = 12.8 Hz, J′ = 6.0 Hz, 2H, 10(13)-Heq], 2.45 product with ethyl acetate to obtain a white solid, mp 214−215 °C. (m, 1H, 8-Ha), 2.48 (m, 1H, 8-Hb), 2.72 (m, 1H, 2′-Hax or 6′-Hax), IR (ATR): 2924, 2851, 1675, 1610, 1546, 1493, 1451, 1361, 1319, 3.05−3.19 (complex signal, 3H, 5(11)-H, 6′-Hax or 2′-Hax), 3.67− 1296, 1282, 1246, 1225, 1208, 1120, 1084, 1017, 991, 946, 908, 874, 3.80 (complex signal, 2H, 4′-H, 6′-Heq or 2′-Heq), 4.41 (dm, J = 13.6 810, 755, 730, 696, 644, 619, 564 cm−1. 1H NMR (400 MHz, Hz, 1H, 2′-Heq or 6′-Heq), 4.78 (d, J = 7.6 Hz, 1H, C4′-NH), 4.85 (s, CDCl3): δ 1.18 [dq, J = 12.0 Hz, J′ = 4.0 Hz, 2H, 3′(5′)-Hax], 1.56 1H, C7−NH), 7.04 [m, 2H, 1(4)-H], 7.09 [m, 2H, 2(3)-H]. 13C [m, 2H, 3″(5″)-Hax], 1.80−1.91 [complex signal, 3H, 3″(5″)-Heq, 3′- NMR (100.6 MHz, CDCl3): δ 21.5 (CH3, COCH3), 32.4 (CH2, C5′ Heq or 5′-Heq], 1.94 [d, J = 13.2 Hz, 2H, 6(12)-Hax], 2.08 (d, J = 12.8 or C3′), 33.7 (CH2, C3′ or C5′), 38.89 (CH2, C6 or C12), 38.96 Hz, 1H, 5′-Heq or 3′-Heq), 2.16 [d, 2H, 10(13)-Hax], 2.20 [m, 2H, 13671 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article 6(12)-Heq] 2.36 [dd, J = 13.2 Hz, J′= 6.4 Hz, 2H, 10(13)-Heq], 2.48 washed with brine (3 mL), dried over anhYd Na2SO4, filtered, and (s, 2H, 8-H), 2.66−2.78 (complex signal, 2H, 4″-H, 6′-Hax or 2′-Hax), evaporated under vacuum to obtain 1−2 mL of a solution of 3.11 (m, 1H, 2′-Hax or 6′-Hax), 3.15 [t, J = 6.0 Hz, 2H, 5(11)-H], 3.43 isocyanate in DCM. To this solution was added (4-aminopiperidin-1- [t, J = 11.6 Hz, 2H, 2″(6″)-Hax], 3.75 (m, 1H, 4′-H), 3.83 (d, J = 13.2 yl) (cyclopropyl)methanone (77 mg, 0.46 mmol). The mixture was Hz, 1H, 2′-Heq or 6′-Heq), 3.99 [dm, J = 11.6 Hz, 2″(6″)-Heq], 4.46 stirred overnight at room temperature, and the solvent was then (m, 1H, 6′-Heq or 2′-Heq), 4.51 (d, J = 7.6 Hz, 1H, C4′-NH), 4.57 (s, evaporated. Column chromatography (SiO2, DCM/methanol mix- 1H, C7−NH), 7.06 [m, 2H, 1(4)-H], 7.09 [m, 2H, 2(3)-H]. 13C tures) provided urea 17 as a white solid (70 mg, 35% yield), mp 119− NMR (100.6 MHz, CDCl3): δ 29.1 [CH2, C3″(5″)], 32.4 (CH2, C5′ 120 °C. IR (ATR): 3367, 3330, 2926, 2853, 1682, 1654, 1605, 1565, or C3′), 34.2 (CH2, C3′ or C5′), 37.6 (CH, C4″), 38.9 [CH2, 1550, 1481, 1452, 1374, 1357, 1319, 1299, 1264, 1224, 1128, 1088, C6(12)], 41.1 (CH2, C6′ or C2′), 41.2 [CH, C5(11)], 44.3 (CH2, 1036, 1013, 993, 967, 948, 925, 911, 870, 799, 755, 735, 700, 632, C2′ or C6′), 44.5 [CH2, C10(13)], 47.0 (CH, C4′), 50.8 (CH2, C8), 604, 564 cm−1. 1H NMR (400 MHz, CDCl3): δ 0.75 (m, 2H, 2″(3″)- 55.6 (C, C7), 67.2 [CH2, C2″(6″)], 69.5 (C, C9), 126.8 [CH, 2(3)], Hax), 0.94 [m, 2H, 2″(3″)-Heq], 1.23 [m, 2H, 3′(5′)-Hax], 1.74 (m, 128.1 [CH, 1(4)], 144.7 [C, C5a(11a)], 156.0 (NHCONH), 172.9 1H, 1″-H), 1.88 [m, 1H, 5′-Heq or 3′-Heq], 1.95 [d, J = 13.2 Hz, 2H, (NCOR). HRMS calcd for [C27H36ClN3O3 + H]+, 486.2518; found, 6(12)-Hax], 2.08 (m, 1H, 3′-Heq or 5′-Heq), 2.16 [d, J = 13.2 Hz, 2H, 486.2522. Anal. Calcd for C27H36ClN3O3: C, 66.72; H, 7.47; N, 8.65. 10(13)-Hax], 2.23 [m, 2H, 6(12)-Heq], 2.37 [dd, J = 12.0 Hz, J′ = 6.4 Found: C, 66.92; H, 7.40; N, 8.43. HPLC: tr = 4.523 (λ = 220 nm, Hz, 2H, 10(13)-Hax], 2.50 (s, 2H, 8-H), 2.73 (broad t, J = 12.0 Hz, 97.1% purity). 1H, 2′-Hax or 6′-Hax), 3.16 [t, J = 6.4 Hz, 2H, 5(11)-H], 3.21 (m, 1H, 4.1.8. 1-(9-Chloro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- 6′-Hax or 2′-Hax), 3.77 (m, 1H, 4′-H), 4.14 (m, 1H, 6′-Heq or 2′-Heq), dimethanobenzo[9]annulen-7-yl)-3-(1-(isopropylsulfonyl)- 4.23 (d, J = 8.0 Hz, 1H, C4′-NH), 4.30 (s, 1H, C7−NH), 4.48 (dm, J piperidin-4-yl)urea (16). To a solution of 9-chloro-5,6,8,9,10,11- = 12.0 Hz, 1H, 3′-Heq or 5′-Heq), 7.05 [m, 2H, 1(4)-H], 7.11 [m, 2H, hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine hydro- 2(3)-H]. 13C NMR (100.6 MHz, CDCl3): δ 7.4 [CH2, C2″(3″)], chloride (268 mg, 0.94 mmol) in DCM (8 mL) and saturated 11.0 (CH, C1″), 32.3 (CH2, C5′ or C3′), 34.1 (CH2, C3′ or C5′), aqueous NaHCO3 solution (5 mL) was added triphosgene (103 mg, 38.9 [CH2, C6(12)], 41.3 [CH, C5(11)], 41.6 (CH2, C2′ or C6′), 0.35 mmol). The biphasic mixture was stirred at room temperature 44.5 [CH2, C10(13)], 44.6 (CH2, C6′ or C2′), 46.7 (CH, C4′), 50.8 for 30 min, then the two phases were separated, and the organic layer (CH2, C8), 55.5 (C, C7), 69.6 (C, C9), 126.8 [CH, C2(3)], 128.1 was washed with brine (5 mL), dried over anhyd Na2SO4, filtered, and [CH, C1(4)], 144.8 (C, C4a(11a)], 156.3 (C, NHCONH), 172.2 (C, evaporated under vacuum to obtain 1−2 mL of a solution of the NCOR). HRMS calcd for [C25H32ClN3O2 + H]+, 442.2256; found, isocyanate in DCM. 442.2262. Anal. Calcd for C25H32ClN3O2·0.75 H2O: C, 66.05; H, To a solution of 1-(isopropylsulfonyl)piperidin-4-amine (194 mg, 7.41; N, 9.24. Found: C, 66.21; H, 7.31; N 9.00. 0.94 mmol) in anhyd THF (8 mL) under an argon atmosphere at 4.1.10. 1-(9-Chloro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- −78 °C was added dropwise a solution of n-butyllithium (2.5 M in dimethanobenzo[9]annulen-7-yl)-3-(1-(2,2,2-trifluoroacetyl)- hexanes, 0.49 mL, 1.22 mmol) for 20 min. After the addition, the piperidin-4-yl)urea (18). To a solution of 9-chloro-5,6,8,9,10,11- mixture was tempered to 0 °C using an ice bath. This solution was hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine hydro- added carefully to the solution of the isocyanate from the previous chloride (130 mg, 0.46 mmol) in DCM (4 mL) and saturated step cooled to 0 °C, under an argon atmosphere. The reaction aqueous NaHCO3 solution (3 mL), triphosgene (50 mg, 0.17 mmol) mixture was stirred at room temperature overnight. Methanol (2 mL) was added. The biphasic mixture was stirred at room temperature for was then added to quench any unreacted n-butyllithium. The solvents 30 min, then the two phases were separated, and the organic one was were evaporated under vacuum to give a yellow residue (690 mg). washed with brine (3 mL), dried over anhyd Na2SO4, filtered, and Column chromatography (SiO2, DCM/methanol mixtures) gave a evaporated under vacuum to obtain 1−2 mL of a solution of white solid. Crystallization from hot DCM/pentane provided urea 16 isocyanate in DCM. To this solution was added 1-(4-aminopiperidin- as a yellowish solid (75 mg, 17% yield). The analytical sample was 1-yl)-2,2,2-trifluoroethan-1-one hydrochloride (106 mg, 0.46 mmol) obtained by crystallization from hot ethyl acetate/pentane mixtures, and Et3N (92 mg, 0.91 mmol). The mixture was stirred overnight at mp 223−224 °C. IR (NaCl disk): 3407, 3370, 2926, 2856, 1672, room temperature, and the mixture was washed with water (15 mL). 1538, 1494, 1451, 1353, 1304, 1296, 1223, 1209, 1177, 1130, 1090, The organic phase was dried over anhyd Na2SO4, filtered, and 1045, 972, 949, 903, 885, 841, 805, 767, 735, 668, 623 cm−1. 1H evaporated under vacuum to obtain an orange gum (196 mg). NMR (400 MHz, CDCl3): δ 1.31 [d, J = 6.8 Hz, 6H, CH(CH3)2], Column chromatography (SiO2, DCM/methanol mixtures) provided 1.37 [dq, J = 12.4 Hz, J′ = 4.0 Hz, 2H, 3′(5′)-Hax], 1.91−1.99 urea 18 as a yellowish solid (55 mg, 26% yield). The analytical sample [complex signal, 4H, 6(12)-Hax, 3′(5′)-Heq], 2.15 [d, J = 13.2 Hz, 2H, was obtained by a crystallization from hot ethyl acetate/pentane 10(13)-Hax], 2.20 [dd, J = 13.6 Hz, J′ = 5.6 Hz, 6(12)-Heq], 2.35 [dd, mixtures, mp 188−189 °C. IR (ATR): 3348, 2926, 2859, 1689, 1634, J = 13.6 Hz, J′ = 5.6 Hz, 10(13)-Heq], 2.47 (s, 2H, 8-H), 2.93 [dt, J = 1556, 1495, 1466, 1454, 1357, 1298, 1266, 1203, 1179, 1137, 1091, 13.2 Hz, J′ = 2.6 Hz, 2H, 2′(6′)-Hax], 3.11−3.22 [complex signal, 3H, 1044, 1009, 992, 971, 946, 897, 802, 757, 698, 660, 623, 599, 556 CH(CH3)2, 5(11)-H], 3.67 (m, 1H, 4′-H), 3.79 [dm, J = 13.2 Hz, cm−1. 1H NMR (400 MHz, CDCl3): δ 1.30 [m, 2H, 5′(3′)-Hax], 1.94 2H, 2′(6′)-Heq], 4.50 (s, 1H, C7−NH), 4.54 (d, J = 7.6 Hz, C4′-NH), [d, J = 12.8 Hz, 2H, 6(12)-Hax], 2.03 [m, 2H, 5′(3′)-Heq], 2.16 [d, J = 7.04 [m, 2H, 1(4)-H], 7.10 [m, 2H, 2(3)-H]. 13C NMR (100.6 MHz, CDCl3): δ 16.7 [CH3, CH(CH3)2], 33.4 [CH2, C3′(5′)], 38.9 [CH2, 13.6 Hz, 2H, 10(13)-Hax], 2.20 [m, 2H, 6(12)-Heq], 2.36 [dd, J = 13.6 C6(12)], 41.2 [CH, C5(11)], 44.5 [CH2, C10(13)], 45.7 [CH2, Hz, J′ = 13.6 Hz, 2H, 10(13)-Heq], 2.47 (s, 2H, 8-H), 2.89 (t, J = 12.0 C2′(6′)], 46.6 (CH, C4′), 50.8 (CH2, C8), 53.4 [CH, CH(CH3)2], Hz, 1H, 2′-Hax or 6′-Hax), 3.13−3.25 [complex signal, 3H, 5(11)-H, 55.6 (C, C7), 69.5 (C, C9), 126.8 [CH, C2(3)], 128.1 [CH, C1(4)], 6′-Hax or 2′-Hax], 3.80 (m, 1H, 4′-H), 3.95 (d, J = 14.0 Hz, 1H, 6′-Heq 144.8 [C, C4a(11a)], 156.0 (C, NHCONH). HRMS calcd for or 2′-Heq), 4.28 (d, J = 7.6 Hz, 1H, C4′-NH), 4.32 (s, 1H, C7−NH), [C24H34ClN3O3S + H]+, 480.2082; found, 480.2084. Anal. Calcd for 4.42 (dm, J = 14.0 Hz, 2′-Heq or 6′-Heq), 7.05 [m, 2H, 1(4)-H], 7.09 C24H34ClN3O3S·0.05 ethyl acetate: C, 60.00; H, 7.16; N, 8.67. Found: [m, 2H, 2(3)-H]. 13C NMR (100.6 MHz, CDCl3): δ 32.2 (CH2, C5′ C, 60.38; H, 7.08; N, 8.27. or C3′), 33.3 (CH2, C3′ or C5′), 38.91 (CH2, C6 or C12), 38.92 4.1.9. 1-(9-Chloro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- (CH2, C12 or C6), 41.2 [CH, C5(11)], 42.8 (CH2, C2′ or C6′), 44.4 dimethanobenzo[9]annulen-7-yl)-3-(1-(cyclopropanecarbonyl)- [CH2, C10(13)], 44.7 (q, 4JC−F = 3.5 Hz, CH2, C6′ or C2′), 46.7 piperidin-4-yl)urea (17). To a solution of 9-chloro-5,6,8,9,10,11- (CH, C4′), 50.8 (CH2, C8), 55.8 (C, C7), 69.3 (C, C9), 116.5 (q, 1 hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine hydro- JC−F = 287.7 Hz, C, CF3), 126.8 [CH, C2(3)], 128.1 [CH, C1(4)], chloride (130 mg, 0.46 mmol) in DCM (4 mL) and saturated 144.7 [C, C4a(11a)], 155.3 (C, NCOR), 155.6 (C, NHCONH). aqueous NaHCO3 solution (3 mL), triphosgene (50 mg, 0.17 mmol) HRMS calcd for [C23H27ClF3N3O2 − H]−, 468.1671; found, was added. The biphasic mixture was stirred at room temperature for 468.1671. Anal. Calcd for C23H27ClF3N3O2·0.75 CH3OH: C, 57.75; 30 min, then the two phases were separated, and the organic one was H, 6.12; N, 8.51. Found: C, 58.04; H, 5.82; N, 8.20. 13672 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article 4.1.11. 1-(9-Chloro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- 45.4 (CH2, C2′ or C6′), 46.6 (CH, C4′), 46.8 (CH2, C8), 56.8 (C, d, dimethanobenzo[9]annulen-7-yl)-3-(1-(1-fluorocyclopropane-1- 3 JC−F = 11.4 Hz, C7), 94.4 (C, d, 1JC−F = 176.9, C9), 126.8 [CH, carbonyl)piperidin-4-yl)urea (19). To a solution of 9-chloro- C2(3)], 128.1 [CH, C1(4)], 144.8 [C, d, 4JC−F = 2.0 Hz, C4a(11a)], 5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7- 156.2 (C, NHCONH), 169.1 (C, COCH3). Anal. Calcd for amine hydrochloride (130 mg, 0.46 mmol) in DCM (4 mL) and C23H30FN3O2·0.5 H2O: C, 67.62; H, 7.65; N, 10.29. Found: C, saturated aqueous NaHCO3 solution (3 mL), triphosgene (50 mg, 67.61; H, 7.93; N, 9.94. HRMS calcd for [C23H30FN3O2 + H]+, 0.17 mmol) was added. The biphasic mixture was stirred at room 400.2395; found, 400.2395. temperature for 30 min, then the two phases were separated, and the 4.1.13. 1-(9-Fluoro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- organic one was washed with brine (3 mL), dried over anhyd Na2SO4, dimethanobenzo[9]annulen-7-yl)-3-(1-(tetrahydro-2H-pyran-4- filtered, and evaporated under vacuum to obtain 1−2 mL of a solution carbonyl)piperidin-4-yl)urea (21). To a solution of 9-fluoro- of isocyanate in DCM. To this solution were added (4-amino- 5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7- piperidin-1-yl) (1-fluorocyclopropyl)methanone hydrochloride (101 amine hydrochloride (150 mg, 0.56 mmol) in DCM (4.5 mL) and mg, 0.46 mmol) and Et3N (92 mg, 0.91 mmol). The mixture was saturated aqueous NaHCO3 solution (3.5 mL), triphosgene (61.5 mg, stirred overnight at room temperature, and the mixture was washed 0.21 mmol) was added. The biphasic mixture was stirred at room with water (10 mL). The organic phase was dried over anhyd Na2SO4, temperature for 30 min, then the two phases were separated, and the filtered, and evaporated under vacuum to obtain an orange gum (140 organic layer was washed with brine (3.5 mL), dried over anhYd mg). Column chromatography (SiO2, DCM/methanol mixtures) Na2SO4, filtered, and evaporated under vacuum to obtain 1−2 mL of provided urea 19 as a yellowish solid (20 mg, 10% yield). The a solution of the isocyanate in DCM. To this solution was added (4- analytical sample was obtained by a crystallization from hot ethyl aminopiperidin-1-yl) (tetrahydro-2H-pyran-4-yl)methanone (119 mg, acetate/pentane mixtures, mp 120−121 °C. IR (ATR): 3340, 2921, 0.56 mmol). The mixture was stirred overnight at room temperature, 2856, 1730, 1632, 1553, 1493, 1453, 1439, 1356, 1327, 1299, 1274, and the solvent was then evaporated. Column chromatography (SiO2, 1244, 1204, 1122, 1088, 1047, 1025, 993, 970, 947, 907, 801, 760, DCM/methanol mixtures) provided urea 21 as a yellowish solid (75 729, 697, 680, 643 cm−1. 1H NMR (400 MHz, CDCl3): δ 1.14−1.38 mg, 28% yield), mp 210−213 °C. IR (ATR): 3351, 2926, 2850, 1609, [complex signal, 6H, 2″(3″)-H2, 5′(3′)-Hax], 1.95 [d, J = 13.2 Hz, 2H, 1549, 1444, 1358, 1306, 1210, 1125, 1089, 1005, 983, 867, 760 cm−1. 6(12)-Hax], 2.00 [m, 2H, 5′(3′)-Heq], 2.16 [d, J = 13.2 Hz, 2H, 1 H NMR (400 MHz, CDCl3): δ 1.17 [dq, 2H, J = 12.0 Hz, J′ = 4.0 10(13)-Hax], 2.22 [dd, J = 12.4 Hz, J′ = 5.6 Hz, 2H, 6(12)-Heq], 2.36 Hz, 3′(5′)-Hax], 1.56 [t, 2H, J = 10.8 Hz, 3″(5″)-Hax], 1.76−2.00 [dd, J = 12.4 Hz, J′ = 6 Hz, 10(13)-Heq], 2.49 (s, 2H, 8-H), 2.83 (m, [complex signal, 7H, 10(13)-Hax, 6(12)-Hax, 3″(5″)-Heq, 3′-Heq or 5′- 1H, 2′-Hax or 6′-Hax), 3.15 (broad signal, 1H, 6′-Hax or 2′-Hax), 3.16 Heq], 2.02−2.20 [complex signal, 5H, 10(13)-Heq, 6(12)-Heq, 5′-Heq [t, J = 6.4 Hz, 2H, 5(11)-H], 3.79 (m, 1H, 4′-H), 4.21 (d, J = 8.0 Hz, or 3′-Heq], 2.21 (d, 2H, J = 6.0 Hz, 8-H2), 2.65−2.80 (complex signal, 1H, C4′-NH), 4.27 (s, 1H, C7−NH), 4.18−4.22 (m, 2H, 2′-Heq, 6′- 2H, 4″-H, 2′-Hax or 6′-Hax), 3.11 (t, 1H, J = 12.4 Hz, 6′-Hax or 2′- Heq), 7.06 [m, 2H, 2(3)-H], 7.10 [m, 2H, 1(4)-H]. 13C NMR (100.6 Hax), 3.21 [broad signal, s, 2H, 5(11)-H], 3.43 [t, 2 H, J = 11.2 Hz, MHz, CDCl3): δ 11.8 [CH2, 2″(3″)-H], 33.6 [CH2, C3′(5′)], 38.9 2″(6″)-Hax], 3.75 (m, 1H, 4′-H), 3.83 (d, 1H, J = 13.2 Hz, 2′-Heq or [CH2, C6(12)], 41.2 [CH, C5(11)], 44.5 [2 CH2, C10(13), 6′-Heq), 3.99 [dd, 2H, J = 11.6 Hz, J′= 2.0 Hz, 2″(6″)-Heq], 4.47 (d, C2′(6′)], 47.2 (CH, C4′), 50.8 (CH2, C8), 55.7 (C, C7), 69.4 (C, 1H, J = 14.0 Hz, 6′-Heq or 2′-Heq), 4.55 (d, 1H, J = 7.6 Hz, C4′-NH), C9), 79.2 (C, C1″), 126.8 [CH, C2(3)], 128.1 [CH, C1(4)], 144.7 4.64 (s, 1H, C7−NH), 7.06 [m, 2H, 1(4)-H], 7.11 [m, 2H, 2(3)-H]. [C, C4a(11a)], 155.7 (C, NHCONH), 166.5 (C, NCOR). HRMS 13 C NMR (100.6 MHz, CDCl3): δ 29.1 [CH2, C3″(5″)], 32.4 (CH2, calcd for [C25H31ClFN3O2 + H]+, 460.2162; found, 460.2165. HPLC: C3′ or 5′), 34.2 (CH2, C5′ or 3′), 37.6 (CH, C4″), 39.3 [CH2, tr = 4.297 (λ = 220 nm, 97.2% purity). C6(12)], 39.5 [CH, 3JC−F = 13.4 Hz, C5(11)], 40.1 [CH2, d, 2JC−F = 4.1.12. 1-(1-Acetylpiperidin-4-yl)-3-(9-fluoro-5,6,8,9,10,11-hexa- 20.1 Hz, C10(13)], 41.1 (CH2, C2′ or 6′), 44.3 (CH2, C2′ or 6′), hydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea (20). To 46.7 (CH2, d, 2JC−F = 17.9 Hz, C8), 46.9 (CH, C4′), 56.9 (C, d, 3JC−F a solution of 9-fluoro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- = 11.5 Hz, C7), 67.1 (CH2, C2″(6″)], 94.4 [C, d, 1JC−F = 177.2 Hz, dimethanobenzo[9]annulen-7-amine hydrochloride (143 mg, 0.53 C9), 126.8 [CH, C1(4)], 128.1 [CH, C2(3)], 144.8 [C, C1′(4′)], mmol) in DCM (4 mL) and saturated aqueous NaHCO3 solution (2 156.0 (C, NHCONH), 172.9 (C, NCOR). Anal. Calcd for mL) was added triphosgene (78 mg, 0.26 mmol). The biphasic C27H36FN3O3·0.2 CH2Cl2: C, 67.14; H, 7.54; N, 8.64. Found: C, mixture was stirred at room temperature for 30 min, then the two 67.47; H, 7.57; N 8.29. HRMS calcd for [C27H36FN3O3 + H], phases were separated, and the organic layer was washed with brine (5 470.2813; found, 470.2815. HPLC: tr = 4.522 (λ = 220 nm, 98.8% mL), dried over anhyd Na2SO4, filtered, and evaporated under purity). vacuum to obtain 1−2 mL of a solution of the isocyanate in DCM. To 4.1.14. 1-(9-Fluoro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- this solution was added 1-(4-aminopiperidin-1-yl)ethan-1-one (90 dimethanobenzo[9]annulen-7-yl)-3-(1-(cyclopropanecarbonyl)- mg, 0.63 mmol). The reaction mixture was stirred at room piperidin-4-yl)urea (22). To a solution of 9-fluoro-5,6,8,9,10,11- temperature overnight, and the solvent was evaporated under vacuum hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-amine hydro- to obtain a yellow gum (259 mg). Column chromatography (SiO2, chloride (150 mg, 0.56 mmol) in DCM (4.5 mL) and saturated DCM/methanol mixtures) gave urea 20 (180 mg, 85% yield). The aqueous NaHCO3 solution (3.5 mL), triphosgene (61.5 mg, 0.21 analytical sample was obtained by crystallization from hot DCM (57 mmol) was added. The biphasic mixture was stirred at room mg), mp 228−229 °C. IR (NaCl disk): 3357, 3063, 3018, 2928, 2857, temperature for 30 min, then the two phases were separated, and the 1684, 1643, 1618, 1553, 1494, 1451, 1359, 1341, 1317, 1267, 1227, organic layer was washed with brine (3.5 mL), dried over anhyd 1207, 1135, 1097, 1042, 1004 cm−1. 1H NMR (400 MHz, CDCl3): δ Na2SO4, filtered, and evaporated under vacuum to obtain 1−2 mL of 1.15 [dq, J = 12.0 Hz, J′ = 4.0 Hz, 1H, 5′-Haxor 3′-Hax], 1.16 (dq, J = a solution of the isocyanate in DCM. To this solution was added (4- 12.0 Hz, J′ = 4.0 Hz, 2H, 3′-Hax or 5′-Hax), 1.83−2.04 [complex aminopiperidin-1-yl) (tetrahydro-2H-pyran-4-yl)methanone (94.2 signal, 6H, 10(13)-Hax, 6-Hax, 12-Hax, 5′-Heq or 3′-Heq], 2.06 (s, 3H, mg, 0.56 mmol). The mixture was stirred overnight at room COCH3), 2.09−2.26 [complex signal, 6H, 8-H2, 10(13)-Heq, 6-Heq, temperature, and the solvent was then evaporated. Column 12-Heq], 2.71 (m, 1H, 6′-Hax or 2′-Hax), 3.12 (m, 1H, 2′-Hax or 6′- chromatography (SiO2, DCM/methanol mixtures) provided urea 22 Hax), 3.21 [t, J = 7.2 Hz, 2H, 5(11)-H], 3.69−3.77 (complex signal, as a white solid (60 mg, 25% yield), mp 187−191 °C. IR (ATR): 2H, 4′-H, 6′-Heq or 2′-Heq), 4.42 (dm, J = 13.6 Hz, 1H, 2′-Heq or 6′- 3320, 2934, 1630, 1568, 1450, 1358, 1317, 1221, 1125, 865, 767, 734, Heq), 4.71 (d, J = 7.6 Hz, 1H, C4′-NH), 4.82 (s, 1H, C7−NH), 7.06 569. 1H NMR (400 MHz, CDCl3): δ 0.75 [dd, 2H, J = 8.0 Hz, J′ = [m, 2H, 1(4)-H], 7.10 [m, 2H, 2(3)-H]. 13C NMR (100.6 MHz, 3.2 Hz, 8′(9′)-Hax], 0.93 [dd, 2H, J = 9.6 Hz, J′ = 4.8 Hz, 8′(9′)-Heq], CDCl3): δ 21.4 (CH3, COCH3), 32.3 (CH2, C5′ or C3′), 33.7 (C3′ 1.20 [complex signal, 2H, 3′(5′)-Hax], 1.74 (tt, 1H, J = 8.0 Hz, J′ = or C5′), 39.3 (CH2, d, 4JC−F = 2.2 Hz, C6 or C12), 39.3 (CH2, d, 4.8 Hz, 7′-H), 1.95−1.85 [d, 4H, J = 12.8 Hz, 10(13)-Hax, 6(12)-Hax; 4 JC−F = 2.2 Hz, C12 or C6), 39.6 [CH, d, 3JC−F = 13.3 Hz, C5(11)], d, 1H, J = 12.4 Hz, 3′ or 5′-Heq], 2.2−2.1 [complex signal, 5H, 40.1 [CH2, d, 2JC−F = 20.1 Hz, C10(13)], 40.7 (CH2, C6′ or C2′), 10(13)-Heq, 6(12)-Heq, 3′ or 5′-Heq], 2.25 (d, 2H, J = 5.2 Hz, 8-H), 13673 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article 2.75 (t, 2H, J = 12 Hz, 2′ or 6′-Hax), 3.20 [m, 3H, 5(11)-H, 2′ or 6′- 5′-Hax or 3′-Hax), 1.79−1.86 [complex signal, 3H, 6(12)-Hax, 5′-Heq Hax], 3.75 (m, 1H, 4′-H), 4.10 (broad signal, d, 1H, J = 14 Hz, 2′ or or 3′-Heq], 1.92 [dm, J = 12.4 Hz, 6(12)-Heq], 1.98−2.02 [complex 6′-Heq), 4.37 (d, 1H, J = 7.6 Hz, HNCONH), 4.50−4.45 (s, 1H, signal, 5H, 10(13)-Hax, 8-H2, 5′-Heq or 3′-Heq], 2.06 (s, 3H, COCH3), HNCONH; s, 1H, 2′ or 6′-Heq), 7.07 [broad signal, 2H, 2(3)-H], 2.10 [m, 2H, 10(13)-Heq], 2. 70 (m, 1H, 6′-Hax or 2′-Hax), 3.10 [ddd, 7.11 [broad signal, 2H, 1(4)-H]. 13C NMR (100.5 MHz, CDCl3): δ J = 14.4 Hz, J′ = 12.4 Hz, J″ = 2.4 Hz, 1H, 2′-Hax or 6′-Hax], 3.17 [t, J 7.51 [CH2, C8′(9′)], 11.15 (CH, C7′), 32.54 (CH2, C5′ or 3′), 34.41 = 6.0 Hz, 2H, 5(11)-H], 3.22 (s, 3H, OCH3), 3.68−3.77 (complex (CH2, C5′ or 3′), 39.47 [CH, C5(11)], 39.80 [CH2, d, 4JC−F = 14.07 signal, 2H, 4′-H, 6′-Heq or 2′-Heq), 4.41 (dm, J = 13.6 Hz, 1H, 2′-Heq Hz, C6(12)], 40.34 [CH2, d, 2JC−F = 20.1 Hz, C10(13), 41.66 (CH2, or 6′-Heq), 4.73 (d, J = 8.0 Hz, 1H, C4′-NH), 4.76 (s, 1H, C7−NH), C2′ or 6′), 44.71 (CH2, C2′ or 6′), 46.94 (CH2, d, 2JC−F = 18.09 Hz, 7.05 [m, 2H, 1(4)-H], 7.08 (m, 2H, 2(3)-H]. 13C NMR (100.6 MHz, C8), 47.22 (CH, C4′), 57.19 (C, C7), 94.53 (C, d, 1JC−F = 176.88 Hz, CDCl3): δ 21.4 (CH3, COCH3), 32.4 (CH2, C5′ or C3′), 33.7 (CH2, C9), 126.99 [CH, C1(4)], 128.27 [CH, C2(3)], 144.97 [CH, C3′ or C5′), 38.2 [CH2, C6(12)], 39.71 (CH2, C10 or C13), 39.73 C1′(4′)], 156.09 (C, HNCONH), 172.26 (C, CO). Anal. Calcd for (CH2, C13 or C10), 39.8 [CH, C5(11)], 40.7 (CH2, C6′ or C2′), C25H32FN3O2·0.1 CH2Cl2: C, 69.46; H, 7.48; N, 9.68. Found: C, 45.4 (CH2, C2′ or C6′), 45.6 (CH2, C8), 46.6 (CH, C4′), 48.2 (CH3, 69.64; H, 7.52; N, 9.45. Accurate mass calcd for [C25H32FN3O2 + OCH3), 55.8 (C, C7), 74.8 (C, C9), 126.6 CH, C2(3)], 128.0 [CH, H]+, 426.2551; found, 426.2556. C1(4)], 145.4 [C, C4a(11a)], 156.3 (C, NHCONH), 169.1 (C, 4.1.15. 1-(1-Acetylpiperidin-4-yl)-3-(9-hydroxy-5,6,8,9,10,11-hex- COCH3). Anal. Calcd for C24H33N3O3: C, 70.04; H, 8.08; N, 10.21. ahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea (23). To Found: C, 69.63; H, 8.28; N, 9.86. HRMS calcd for [C24H33N3O3 a solution of 1-(4-aminopiperidin-1-yl)ethan-1-one (192 mg, 1.35 +H]+, 412.2595; found, 412.2595. mmol) in DCM (4 mL) and saturated aqueous NaHCO3 solution (3 4.1.17. 1-(5,6,8,9,10,11-Hexahydro-7H-5,9:7,11- mL), triphosgene (200 mg, 0.67 mmol) was added. The biphasic dimethanobenzo[9]annulen-7-yl-9-d)-3-(1-(tetrahydro-2H-pyran- mixture was stirred at room temperature for 30 min, then the two 4-carbonyl)piperidin-4-yl)urea (25). To a solution of 5,6,8,9,10,11- phases were separated, and the organic one was washed with brine (5 hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-9-d-7-amine hy- mL), dried over anhyd Na2SO4, filtered, and evaporated under drochloride (82 mg, 0.32 mmol) in DCM (2 mL) and saturated vacuum to obtain 1−2 mL of a solution of the isocyanate in DCM. To aqueous NaHCO3 solution (2 mL), triphosgene (36 mg, 0.12 mmol) this solution was added 9-amino-5,6,8,9,10,11-hexahydro-7H- was added. The biphasic mixture was stirred at room temperature for 5,9:7,11-dimethanobenzo[9]annulen-7-ol hydrochloride (300 mg, 30 min, then the two phases were separated, and the organic one was 1.13 mmol) followed by Et3N (228 mg, 2.25 mmol). The reaction washed with brine (3 mL), dried over anhyd Na2SO4, filtered, and mixture was stirred at room temperature overnight, and the solvent evaporated under vacuum to obtain 1−2 mL of a solution of was evaporated under vacuum. Column chromatography (SiO2, isocyanate in DCM. To this solution was added (4-aminopiperidin-1- DCM/methanol mixtures) gave urea 23 (19 mg, 4.2% yield) as a gray yl) (tetrahydro-2H-pyran-4-yl)methanone (68 mg, 0.32 mmol). The solid, mp 222−223 °C. IR (NaCl disk): 3313, 2922, 2852, 1733, mixture was stirred overnight at room temperature, and the solvent 1716, 1699, 1646, 1622, 1558, 1542, 1507, 1491, 1472, 1456, 1358, was then evaporated. Column chromatography (SiO2, DCM/ 1337, 1319, 1301, 1265, 1231, 1204, 1134, 1104, 1053 cm−1. 1H Methanol mixtures) provided urea 25 as a white solid (83 mg, 56% NMR (400 MHz, CDCl3): δ 1.20 [m, 2H, 3′(5′)-Hax], 1.76 [d, J = yield). The analytical sample was obtained by crystallization from hot 12.8 Hz, 2H, 6(12)-Hax], 1.86−2.02 [complex signal, 6H, 3′(5′)-Heq, EtOAc, mp 125−126 °C. IR (ATR): 3318, 2902, 2849, 1630, 1557, 10(13)-Hax, 6(12)-Heq], 2.04 (s, 2H, 8-H), 2.07 (3, 3H, COCH3), 1491, 1445, 1361, 1318, 1300, 1274, 1238, 1213, 1123, 1108, 1090, 2.14 [m, 2H, 10(12)-Heq], 2.70 (m, 1H, 6′-Hax or 2′-Hax), 3.12 [ddd, 1040, 1016, 987, 972, 872, 823, 750 cm−1. 1H NMR (400 MHz, J = 14.4 Hz, J′ = 12.0 Hz, J″ = 2.4 Hz, 1H, 2′-Hax or 6′-Hax], 3.17 [t, J CDCl3): δ 1.17 [dt, J = 12.0 Hz, J′ = 4.0 Hz, 2H, 3′-Hax or 5′-Hax], = 6.0 Hz, 2H, 5(11)-H], 3.67−3.78 [complex signal, 2H, 4′-H, 2′-Heq 1.20 [dt, J = 12.0 Hz, J′ = 4.0 Hz, 2H, 5′-Hax or 3′-Hax], 1.56 [m, 2H, or 6′-Heq], 4.24 (d, J = 7.6 Hz, 1H, C4′-NH), 4.34 (s, 1H, C7−NH), 3″(5″)-Hax], 1.73 [d, J = 13.2 Hz, 2H, 10(13)-Hax], 1.80−1.90 4.47 [dm, J = 14.0 Hz, 1H, 6′-Heq or 2′-Heq), 7.06 [m, 2H, 1(4)-H)], [complex signal, 3H, 3″(5″)-Heq, 5′-Heq or 3′-Heq), 1.92 [dd, J = 13.2 7.09 [m, 2H, 2(3)-H]. 13C NMR (100.6 MHz, CDCl3): δ 21.4 (CH3, Hz, J′ = 6.0 Hz, 2H, 10(13)-Heq], 1.98−2.10 [complex signal, 5H, COCH3), 32.4 (CH2, C5′ or C3′), 33.6 (CH2, C3′ or C5′), 39.4 6(12)-Hax, 8-H2, 3′-Heq or 5′-Heq], 2.18 [m, 2H, 6(12)-Heq], 2.65− [CH2, C10(13)], 40.1 [CH, C5(11)], 40.7 (CH2, C6′ or C2′), 42.5 2.76 [complex signal, 2H, 4″-H, 2′-Hax or 6′-Hax], 3.03 [t, J = 6.0 Hz, [CH2, C6(12)], 45.4 (CH2, C2′ or C6′), 47.1 (CH, C4′), 49.1 (CH2, 2H, 5(11)-H], 3.10 (m, 1H, 6′-Hax or 2′-Hax), 3.43 [m, 2H, 2″(6″)- C8), 56.3 (C, C7), 71.0 (C, C9), 126.6 [CH, C2(3)], 128.1 [CH, Hax], 3.75 (m, 1H, 4′-H), 3.82 (d, J = 13.0 Hz, 1H, 6′-Heq or 2′-Heq), C1(4)], 145.2 [C, C4a(11a)], 155.9 (C, NHCONH), 169.0 (C, 3.99 [dm, J = 11.4 Hz, 2H, 2″(6″)-Heq], 4.27−4.34 [complex signal, COCH3). Anal. Calcd for C23H31N3O3·CH3OH: C, 67.11; H, 8.21; 2H, C7−NH, C4′-NH], 4.48 [d, J = 13.0 Hz, 1H, 2′-Heq or 6′-Heq], N, 9.78. Found: C, 67.25; H, 8.15; N, 9.72. HRMS calcd for 7.03 [m, 2H, 1(4)-H], 7.05 [m, 2H, 2(3)-H]. 13C NMR (100.6 MHz, [C23H31N3O3 + H]+, 398.2438; found, 398.2440. CDCl3): δ 29.2 [CH2, C3″(5″)], 30.7 (CD, t, 1JC−D = 19.8 Hz, C9), 4.1.16. 1-(1-Acetylpiperidin-4-yl)-3-(9-methoxy-5,6,8,9,10,11- 32.4 (CH2, C5′ or C3′), 34.1 (CH2, C3′ or C5′), 34.3 [CH2, hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7-yl)urea C10(13)], 37.6 (CH, C4″), 40.5 [CH2, C6(12)], 41.1 [CH2, C2′ or (24). To a solution of 9-methoxy-5,6,8,9,10,11-hexahydro-7H- C6′), 41.2 [CH, C5(11) and CH2, C8], 44.3 (CH2, C6′ or C2′), 47.1 5,9:7,11-dimethanobenzo[9]annulen-7-amine (300 mg, 1.23 mmol) (CH, C4′), 51.9 (C, C7), 67.2 [CH2, C2″(6″)], 126.2 [CH, C2(3)], in DCM (4.5 mL) and saturated aqueous NaHCO3 solution (3 mL), 128.0 [CH, C1(4)], 146.6 [C, C4a(11a)], 156.1 (C, NHCONH), triphosgene (183 mg, 0.62 mmol) was added. The biphasic mixture 172.8 (C, NCOR). HRMS calcd for [C27H36DN3O3 + H]+, 453.297; was stirred at room temperature for 30 min, then the two phases were found, 453.2974. Anal. Calcd for C27H36DN3O3·1 H2O: C, 68.91; H, separated, and organics were washed with brine (5 mL), dried over 8.14; N, 8.93. Found: C, 69.28; H, 7.94; N 8.69. anhyd Na2SO4, filtered, and evaporated under vacuum to obtain 1−2 4.1.18. tert-Butyl 4-(3-(9-chloro-5,6,8,9,10,11-hexahydro-7H- mL of a solution of the isocyanate in DCM. To this solution was 5,9:7,11-dimethanobenzo[9]annulen-7-yl)ureido)piperidine-1-car- added 1-(4-aminopiperidin-1-yl)ethan-1-one (210 mg, 1.47 mmol). boxylate (26). To a solution of 9-chloro-5,6,8,9,10,11-hexahydro-7H- The reaction mixture was stirred at room temperature overnight, and 5,9:7,11-dimethanobenzo[9]annulen-7-amine hydrochloride (131 mg, the solvent was evaporated under vacuum to obtain a white gum (521 0.46 mmol) in DCM (4 mL) and saturated aqueous NaHCO3 mg). Column chromatography (SiO2, DCM/methanol mixtures) gave solution (3 mL), triphosgene (50 mg, 0.17 mmol) was added. The urea 24 (148 mg, 30% yield) as a white solid. The analytical sample biphasic mixture was stirred at room temperature for 30 min, then the was obtained by crystallization from hot EtOAc, mp 212−213 °C. IR two phases were separated, and the organic one was washed with (NaCl disk): 3358, 3044, 3019, 2931, 2847, 2823, 1646, 1618, 1555, brine (5 mL), dried over anhyd Na2SO4, filtered, and evaporated 1495, 1452, 1356, 1319, 1266, 1229, 1135, 1095, 1076, 972, 849, 756, under vacuum to obtain 1−2 mL of a solution of isocyanate in DCM. 735 cm−1. 1H NMR (400 MHz, CDCl3): δ 1.12 (dq, J = 11.6 Hz, J′ = To this solution was added 4-amino-1-Boc-piperidine (93 mg, 0.46 4.0 Hz, 1H, 3′-Hax or 5′-Hax), 1.19 (dq, J = 11.6 Hz, J′ = 4.0 Hz, 1H, mmol). The mixture was stirred overnight at room temperature, and 13674 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article the solvent was then evaporated. Column chromatography (SiO2, of histidine residues: HIE146, HIE239, HIP251, HID265, HIP334, DCM/Methanol mixtures) provided 26 as a white solid (103 mg, HIE420, HIE506, HIE513, HIE518, and HIP524. 47% yield). HRMS−ESI− m/z: [M − H]− calcd for [C26H36ClN3O3 Each system was immersed in a pre-equilibrated truncated − H]−, 472.2372; found, 472.2365. octahedral box of water molecules with an internal offset distance 4.1.19. 1-(9-Chloro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- of 10 Å. All systems were neutralized with explicit counterions (Na+ dimethanobenzo[9]annulen-7-yl)-3-(piperidin-4-yl)urea (27). t- or Cl−). A two-stage geometry optimization approach was performed. Butyl 4-(3-(9-chloro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- First, a short minimization of the positions of water molecules with dimethanobenzo[9]annulen-7-yl)ureido)piperidine-1-carboxylate positional restraints on the solute by a harmonic potential with a force (19.74 mg, 41.6 μmol) was dissolved in 5 mL dichloromethane and constant of 500 kcal mol−1 Å−2 was done. The second stage was an 20 μL of TFA was added and the reaction stirred until TLC showed unrestrained minimization of all the atoms in the simulation cell. no starting material. The solvents were evaporated, and the free amine Then, the systems were gently heated in six 50 ps steps, increasing the 27 (ESI−MS calcd for C21H28ClN3O [M + H+] m/z: 374.19; found, temperature by 50 K each step (0−300 K) under constant-volume, 374.05) was used in the next step. periodic-boundary conditions, and the particle-mesh Ewald ap- 4.1.20. 1-(1-(3-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)propanoyl)- proach60 to introduce long-range electrostatic effects. For these piperidin-4-yl)-3-(9-chloro-5,6,8,9,10,11-hexahydro-7H-5,9:7,11- steps, a 10 Å cutoff was applied to Lennard−Jones and electrostatic dimethanobenzo[9]annulen-7-yl)urea (28). 1-(9-Chloro- interactions. Bonds involving hydrogen were constrained with the 5,6,8,9,10,11-hexahydro-7H-5,9:7,11-dimethanobenzo[9]annulen-7- SHAKE algorithm.61 Harmonic restraints of 10 kcal mol−1 were yl)-3-(piperidin-4-yl)urea (41.6 μmol, 1.2 equiv) was dissolved in 250 applied to the solute, and the Langevin equilibration scheme was used μL dimethylformamide and 20 μL N,N-diisopropylethylamine was to control and equalize the temperature.62 The time step was kept at 2 added. In a separate flask 3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)- fs during the heating stages, allowing potential inhomogeneities to propanoic acid (34.6 μmol, 1 equiv) was dissolved in 250 μL self-adjust. Each system was then equilibrated for 2 ns with a 2 fs dimethylformamide to which 1-[bis(dimethylamino)methylene]-1H- timestep at a constant pressure of 1 atm (NPT ensemble). Finally, 1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU; conventional MD trajectories at a constant volume and temperature 34 μmol, 0.9 equiv) and N,N-diisopropylethylamine (20 μL, 3 equiv) (300 K) were collected. In total, we carried out three replicas of 500 were added. After stirring for 10 min, the solution containing the ns MD simulations for sEH in the presence of 13, 15, 21, 23, and 24 amine was added to the mixture, which was left stirring at room gathering a total of 7.5 μs of MD simulation time. Each MD temperature for 16 h. Reverse phase HPLC purification provided 28 simulation was clusterized based on active site residues, and the in a 66% yield. ESI−MS calcd for C29H36ClN5O2 [M + H]+ m/z: structures corresponding to the most populated clusters were used for 522.26; found, 522.10. 1H NMR (CDCl3): δ (ppm) 1.18−1.28 (m, the noncovalent interactions analysis. We monitored the presence of 3H), 1.66 (td, 2H), 1.84 (m, 2H), 1.18−2.06 (cs, 12H), 1.16 (m, water molecules using the watershell function of the cpptraj MD 4H), 2.23 (d, 2H), 2.71 (dt, 1H), 3.06 (dt, 1H), 3.23 (s, 2H), 3.69− analysis program.63 aMD simulations64,65 were used to study the 3.74 (cs, 2H), 4.47 (d, 2H), 7.06−7.09 (m, 2H), 7.11−7.14 (m, 2H). spontaneous binding of 15 in the active site of sEH. Standard dual- 13 C NMR (CDCl3): δ (ppm) 13.3, 26.8, 27.9, 32.3, 32.5, 33.3, 39.3, boost aMD simulations were performed using the same simulation 39.5, 39.6, 39.9, 40.1, 40.9, 44.3, 46.6, 46.7, 47.4, 57.2, 57.3, 69.2, protocols and aMD parameters as described in our previous works.21 82.8, 93.7, 94.9, 126.9, 128.2, 144.7, 155.8, 169.5. To reconstruct the spontaneous binding process, we placed one 4.2. In Vitro Biological Methods. The assays for the in vitro molecule of 15 in the solvent with a minimum distance of 25 Å from determination of the inhibitory activities toward human and mouse catalytic Asp335. First, we performed 250 ns of conventional MD sEH, 30 the assays for determination of the PAMPA−BBB followed by 10 replicas of 2 μs of aMD capturing one binding event permeability,36 aqueous solubility, cytotoxicity in SH-SY5Y cells, (see Movie S1 comprising only the aMD simulation part). Binding microsomal stability, cytochrome P450 inhibition, permeability, affinities (kcal/mol) of compounds 13, 15, 21, and 23 were computed hERG inhibition, and inhibition of human LOX-5 were carried out using the MM/GBSA method as implemented in AMBER 18. following described methodologies previously used in our group (see 4.4. Preparation of HEK 293T Lysates. HEK293T cells were the Supporting Information for complete details).21,22 grown in DMEM media (D6546-500ML Sigma) supplemented with 4.3. In Silico Studies. 4.3.1. MD Simulation Details. The 10% FBS, 2 mM glutamax, 100 units/mL penicillin, and 0.1 mg/mL parameters for 13, 15, 21, and 23 for the MD simulations were streptomycin. They were maintained at 37 °C with 5% CO2. Cells generated using the ANTECHAMBER module of AMBER 1851 using were split every 3 to 4 days according to an ATCC protocol. The cells the general AMBER force field (GAFF),52 with partial charges set to were harvested and collected by centrifugation (500 g for 5 min at 4 fit the electrostatic potential generated at the HF/6-31G(d) level by °C) and the supernatant was removed. The pellets were washed twice the RESP model.53 The charges were calculated according to the with ice-cold PBS and resuspended in 2 vol of ice-cold lysis buffer (50 Merz−Singh−Kollman scheme54,55 using Gaussian 09.56 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT and 0.5% NP-40). MD simulations of sEH were carried out using PDB 5AM3 After 30 min on ice, the cells were centrifuged to remove cell debris (crystallized with t-AUCB) and PDB 5ALZ (crystallized with for 5 min at 4 °C. The supernatant was aliquoted and flash frozen in piperidine-based sEHI) as a starting point.31 The benzohomoadama- liquid N2 for use as lysates, with a total protein concentration of 1 natane derivatives corresponding to 13, 15, 21, 23, and 24 were mg/mL. Protein concentrations were determined using the BCA assay manually prepared using the t-AUCB structure as a starting point. (Fisher Scientific). Molecular docking calculations using the standard parameters of the 4.5. Labeling in HEK 293T Lysates. HEK 293T lysates were SwissDock web server were carried out to assess to generate starting spiked or not with 100 ng of recombinant purified sEH, treated either orientations of 13, 15, 21, 23, and 24.57,58 The coordinates of t- with 100 nM probe 28 or DMSO, and incubated for 30 min at 37 °C. AUCB in PDB 5AM3 and piperidine-base compound in PDB 5ALZ After this time, the samples were irradiated for 6 min at 365 nm using were a reference for placing compounds 13, 15, 21, 23, and 24 in a 100 W UV lamp. Subsequently, a bi-functional tag containing a molecular docking calculations in the two possible orientations: (a) TAMRA dye and a biotin was incorporated using copper(I)-catalyzed benzohomoadamantane group in the lhs while the piperidine group in azide−alkyne cycloaddition (CuAAC). The photoaffinity labeling was the rhs and (b) benzohomoadamantane group in the rhs while analyzed by in-gel analysis, mixing the samples with 4× SDS-loading piperidine in the lhs. From these two sets of orientations, buffer, and separating using 12% SDS-PAGE after which the gel was conventional MD simulations were used to explore the conforma- scanned on a Typhoon FLA 9500. tional plasticity of sEH in the presence of 13, 15, 21, 23, and 24 4.6. Labeling Purified Soluble Epoxide Hydrolase for bound in the active site. All simulations were performed using the Minimal Probe Concentration Determination. Purified recombi- AMBER ff14SB force field.59 Amino acid protonation states were nant sEH was produced and purified as indicated previously.30 Of the predicted using the H++ server (http://biophysics.cs.vt.edu/H++). pure active enzyme 100 or 200 ng were incubated for 30 min at 37 °C The MD simulations have been carried with the following protonation with decreasing concentrations of probe 3, namely: 10 μM, 1 μM, 100 13675 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article nM, 10 nM, and 1 nM. After this time, the compounds were irradiated The plasma was separated by centrifugation for 10 min and stored at for 6 min at 365 nm using a 100 W UV lamp. Subsequently, a bi- −80 °C until analysis by HPLC. Frozen plasma samples were thawed functional tag containing a TAMRA dye and a biotin was at room temperature and 25 μL of acetonitrile were added to a 100 incorporated using copper(I)-catalyzed azide−alkyne cycloaddition μL of plasma sample. The sample was vortexed for 30 s and (CuAAC). The photoaffinity labeling was analyzed by in-gel analysis centrifuged (14,000 rpm/min) for 5 min. The supernatant was by mixing the samples with 4× SDS-loading buffer and separating transferred to an injection bottle and 25 μL was injected into the using 12% SDS-PAGE after which the gel was scanned on a Typhoon chromatographic system. FLA 9500. 4.9.3. Instruments and Analysis Conditions. The HPLC system 4.7. EPHX2 Target Engagement Confirmation and Off- was a PerkinElmer LC (PerkinElmer INC, Massachusetts, U.S.) Target Elucidation by Pull Down. Untreated HEK293T whole cell consisting of a Flexar LC pump, a chromatography interface (NCI lysates were normalized to a concentration of 1 mg/mL in a volume of 900 network), a Flexar LC autosampler PE, and a Waters 2487 dual λ 100 μL, per condition. Lysates were then treated with DMSO, 10 μM absorbance detector. The chromatographic column was a kromasil of probe 28 or 10 μM of probe 28, and 100 μM of 15 (for 100-5-C18 (4,0 × 200 mm-Teknokroma Analitica S.A. Sant Cugat, competition experiments), and incubated at 37 °C for 30 min. After Spain). Flow was 0.8 mL/min and the mobile phase consisting in 0.05 this time, the whole was irradiated for 6 min at 365 nm using a 100 W M KH2PO4 (30%)/acetonitrile (70%) in isocratic conditions. The UV lamp. Subsequently, a bi-functional tag containing a TAMRA dye elution times of 15 and 21 were 5.6 and 4.4 min, respectively. and a biotin was incorporated via CuAAC. The excess reagents from Compounds were detected at 220 nm. The assay had a range of the samples were then removed by acetone precipitation. Following 0.015−25 μg mL−1. The calibration curves were constructed by resuspension of the pellets to a final volume of 100 μL, half of the plotting the peak area ratio of analyzed peak against known sample was kept as the input control. The remaining 50 μL were concentrations. Compound 22 was analyzed under the same incubated with 20 μL of pre-washed streptavidin beads (Thermo chromatographic conditions but the response to the analysis was 10 Fisher) for 1 h with mixing at RT. The supernatant was removed, and times lower than that of 15 and 21. the beads were sequentially washed with 0.33% SDS in PBS (2 × 50 4.9.4. Pharmacokinetic Analysis. 15 and 51 plasma concentrations μL), 1 M NaCl (2 × 50 μL) and PBS (2 × 50 μL). Bound proteins versus time curves for the means of animals were analyzed by a non- were eluted by boiling (95 °C) the beads with 60 μL of 1× SDS compartmental model based on the statistical moment theory using loading buffer for 10 min. Samples were resolved by 12% SDS-PAGE. the “PK Solutions” computer program. The pharmacokinetic Following visualization using a Typhoon FLA 9500, the gel was parameters calculated were as follows: area under the plot of plasma transferred onto a nitrocellulose membrane and probed with VEGF2 concentration versus time curve (AUC), calculated using the (cell signaling), p38 MAPK (cell signaling), EPHX1 (Elabscience), trapezoidal rule in the interval 0−6 h; HL (t1/2β), determined as and EPHX2 (Abcam) for detection. ln2/β, being β, calculated from the slope of the linear, least-squares This experiment was also carried out using lower probe and parent regression line; Cmax and Tmax were read directly from the mean compound concentrations of 1 and 10 μM, respectively, yielding the concentration curves. same results. 4.10. In Vivo Efficacy Studies. 4.10.1. Experimental Animals. 4.8. Affinity-Based Probe and Parent Compound Off-Target Experiments were performed in female WT-CD1 (Charles River, Profile Elucidation. To HEK293T cell lysates at 1 mg/mL protein Barcelona, Spain) mice weighing 25−30 g. Mice were acclimated in concentration spiked or not with 100 ng of recombinant human sEH our animal facilities for at least 1 week before testing and were housed and 100 ng of purified recombinant enzyme were treated with either in a room under controlled environmental conditions: 12/12 h day/ 100 nM probe 28, 10 μM urea 15, and 100 nM probe 28 or DMSO to night cycle, constant temperature (22 ± 2 °C), air replacement every a concentration of 1% of the total sample. After 30 min of incubation 20 min, and they were fed a standard laboratory diet (Harlan Teklad of the compounds at 37 °C, the whole was irradiated for 6 min at 365 Research Diet, Madison, WI, USA) and tap water ad libitum until the nm using a 100 W UV lamp. Subsequently, a bi-functional tag beginning of the experiments. The behavioral test was conducted containing a TAMRA dye and a biotin was incorporated via CuAAC. during the light phase (from 9.00 to 15.00 h), and randomly The samples were analyzed by in-gel analysis by mixing the samples throughout the oestrous cycle. Animal care was in accordance with with 4× SDS-loading buffer and separating using 12% SDS-PAGE institutional (Research Ethics Committee of the University of after which the gel was scanned on a Typhoon FLA 9500 and/or Granada, Spain), regional (Junta de Andalucia,́ Spain), and interna- submitted to Western blot analysis using human sEH antibody for tional standards (European Communities Council Directive 2010/ detection (Abcam). The comparison of labeling patterns via 63). fluorescence showed the inability of the parent compound to compete 4.10.2. Drugs and Drug Administration. The sEHI were dissolved out the probe 28 for most of the targets, which pointed out that in 5% DMSO (Merck KGaA, Darmstadt, Germany) in physiological except for the sEH the other labeled proteins are not targets of the sterile saline (0.9% NaCl). Drug solutions were prepared immediately parent compound but of the probe 28. before the start of the experiments and injected sc in a volume of 5 4.9. Pharmacokinetic Study. 4.9.1. Animals. For pharmacoki- mL/kg into the interscapular area. To test for the effects of MS- netic studies, 48 male CD1 mice (weight, 40 to 50 g; age, 8 week), PPOH (Cayman Chemical Company, Ann Arbor, MI, USA), a obtained from Envugi, Madison, WI, USA, were housed 3 per cage in selective inhibitor of microsomal CYP450 epoxidase,45 on the effects IVC (Thoren no. 9 cages (19.5 cm × 30.9 cm × 13.3 cm); Thoren induced by the sEHI tested, this compound was dissolved in DMSO Caging Systems, Hazleton, PA. Mice were kept in an environmentally 5% and cyclodextrin 40% in saline and administered 5 min before controlled room: air replacement every 10 min, constant temperature sEHI injection. When the effect of the association of several drugs was (21 ± 3 °C), and humidity and on a 12/12 h day/night cycle. All assessed, each injection was performed in different areas of the experimental procedures followed the standard ethical guidelines of interscapular zone to avoid the mixture of the drug solutions and any European Communities Council Directive 86/609/EEC and by the physicochemical interaction between them. In all cases, the Institutional Animal Care and Use of Catalunya (10291, approved 1/ researchers who performed the experiments were blinded to the 28/2018). treatment received by each animal. 4.9.2. Drug Delivery, Sample Collection, and Sample Prepara- As it will be detailed below, we used two different algogenic tion. Formulations were prepared the day of the study. The vehicle substances to explore the effects of sEHI on nociception: capsaicin was 10% of 2-hydroxypropyl-β-cyclodextrin (Sigma-Aldrich, ref. was used to induce somatic mechanical hypersensitivity, and CTX to 332607-25G). Mice were administered with 15 or 21 at a dose of 5 induce visceral pain. Capsaicin (Sigma-Aldrich Quimica ́ S.A.) was mg/Kg by sc route. The volume of administration was 10 mL/kg. dissolved in 1% DMSO in physiological sterile saline to a Animals were weighed before each administration to adjust the concentration of 0.05 μg/μL (i.e., 1 μg per mouse). Capsaicin required volume. Blood samples (n = 3) were collected after solution was injected intraplantarly (i.pl.) into the right hind paw euthanasia of mice at different times (0.25; 0.5; 1; 2; 3, 4, and 6 h). proximate to the heel, in a volume of 20 μL using a 1710 TLL 13676 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Hamilton microsyringe (Teknokroma, Barcelona, Spain) with a 301/2- PDB files corresponding to the most populated cluster gauge needle. Control animals were injected with the same volume of obtained from the MD simulations of compounds 15 the vehicle of capsaicin. CTX (Sigma-Aldrich, Madrid, Spain), which (PDB) was used to induce a painful cystitis, was dissolved in saline and PDB files corresponding to the most populated cluster injected ip at a dose of 300 mg/kg, in a volume of 10 ml/kg. The same volume of solvents was injected in control animals. obtained from the MD simulations of compounds 21 4.10.3. Evaluation of Capsaicin-Induced Secondary Mechanical (PDB) Hypersensitivity. Animals were placed into individual test compart- PDB files corresponding to the most populated cluster ments for 2 h before the test to habituate them to the test conditions. obtained from the MD simulations of compounds 13 The test compartments had black walls and were situated on an (PDB) elevated mesh-bottomed platform with a 0.5 cm2 grid to provide PDB files corresponding to the most populated cluster access to the ventral surface of the hind paws. In all experiments, obtained from the MD simulations of compounds 23 punctate mechanical stimulation was applied with a dynamic plantar aesthesiometer (Ugo Basile, Varese, Italy) at 15 min after the (PDB) administration of capsaicin or its solvent. Briefly, a nonflexible PDB files corresponding to the most populated cluster filament (0.5 mm diameter) was electronically driven into the ventral obtained from the MD simulations of compounds 24 side of the paw previously injected with capsaicin or solvent (i.e., the (PDB) right hind paw), at least 5 mm away from the site of the injection Molecular movie of the spontaneous binding accelerated toward the fingers. The intensity of the stimulation was fixed at 0.5 g molecular dynamics simulation of compound 15 into the force, as described previously.66 When a paw withdrawal response sEH active site (MPG) occurred, the stimulus was automatically terminated, and the response Molecular formula string and data (CSV) latency time was automatically recorded. The filament was applied three times, separated by intervals of 0.5 min, and the mean value of the three trials was considered the withdrawal latency time of the animal. A cutoff time of 50 s was used. The compounds tested, or ■ AUTHOR INFORMATION Corresponding Author their solvent, were administered sc 30 min before the i.pl. Santiago Vázquez − Laboratori de Química Farmacèutica administration of capsaicin or DMSO 1% (i.e., 45 min before we (Unitat Associada al CSIC), Facultat de Farmàcia i Ciències evaluated the response to the mechanical punctate stimulus). 4.10.4. Evaluation of Cyclophosphamide-Induced Visceral Pain. de l’Alimentació, and Institute of Biomedicine (IBUB), CTX-evoked pain behaviors and referred hyperalgesia were examined Universitat de Barcelona, Barcelona 08028, Spain; following a previously described protocol with slight modifications.48 orcid.org/0000-0002-9296-6026; Phone: +34 Animals were placed into the same individual test compartments 934024533; Email: svazquez@ub.edu described above for 40 min to habituate them to the test conditions. Then, mice were injected ip with CTX or saline. Compound 21 or its Authors solvent was sc injected at 120 min after CTX ip administration, and Sandra Codony − Laboratori de Química Farmacèutica pain behaviors were recorded for 2 min every 30 min in the period (Unitat Associada al CSIC), Facultat de Farmàcia i Ciències from 150 to 240 min. These pain-related behaviors were coded de l’Alimentació, and Institute of Biomedicine (IBUB), according to the following scale: 0 = normal, 1 = piloerection, 2 = Universitat de Barcelona, Barcelona 08028, Spain labored breathing, 3 = licking of the abdomen, and 4 = stretching and José M. Entrena − Animal Behavior Research Unit, Scientific contractions of the abdomen. At the end of the 2 h observation period Instrumentation Center, Parque Tecnológico de Ciencias de la (i.e., 4 h after the CTX injection), the sensory threshold in the abdomen was measured 240 min after CTX administration, using a Salud, University of Granada, Granada 18100, Spain series of von Frey filaments with bending forces ranging from 0.02 to Carla Calvó-Tusell − CompBioLab Group, Departament de 2 g (Stoelting, Wood Dale, USA). Testing was always initiated with Química and Institut de Química Computacional i Catàlisi the 0.4 g filament. The response to the filament was considered (IQCC), Universitat de Girona, Girona 17003, Spain; positive if immediate licking/scratching of the application site, sharp orcid.org/0000-0003-2681-8460 retraction of the abdomen, or jumping was observed. If there was a Beatrice Jora − Laboratori de Química Farmacèutica (Unitat positive response, a weaker filament was used; if there was no Associada al CSIC), Facultat de Farmàcia i Ciències de response, a stronger stimulus was then selected. The 50% threshold l’Alimentació, and Institute of Biomedicine (IBUB), withdrawal was determined using the up and down methods and Universitat de Barcelona, Barcelona 08028, Spain calculated using the Up−Down Reader software.67 Rafael González-Cano − Department of Pharmacology, ■ * ASSOCIATED CONTENT sı Supporting Information Faculty of Medicine and Biomedical Research Center (Neurosciences Institute), Biosanitary Research Institute ibs.GRANADA, University of Granada, Granada 18016, The Supporting Information is available free of charge at Spain https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c00515. Sílvia Osuna − CompBioLab Group, Departament de Química and Institut de Química Computacional i Catàlisi (IQCC), Complete details of in vitro biological methods, 1H and Universitat de Girona, Girona 17003, Spain; Institució 13 C NMR spectra and elemental analysis data of the new Catalana de Recerca i Estudis Avançats (ICREA), Barcelona compounds, HPLC trace for compounds 15, 19, and 21, 08010, Spain; orcid.org/0000-0003-3657-6469 non-covalent interactions between the inhibitors and the Rubén Corpas − Institute of Biomedical Research of Barcelona active site residues of sEH, analysis of active site water (IIBB), CSIC and IDIBAPS, Barcelona 08036, Spain molecules in MD simulations, plots of the dihedral angle Christophe Morisseau − Department of Entomology and that describes the rotation of the benzohomoadaman- Nematology and Comprehensive Cancer Center, University of tane in the left-hand side pocket of the sEH active site California, Davis, California 95616, United States along the MD simulations, cytochrome inhibition of Belén Pérez − Department of Pharmacology, Therapeutics and compounds 15, 21, and 22, and pharmacokinetic data of Toxicology, Institute of Neurosciences, Autonomous compounds 15 and 21 (PDF) University of Barcelona, Barcelona 08193, Spain 13677 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Marta Barniol-Xicota − Laboratory of Chemical Biology, studies. C.P. and M.I.R.-F. performed the hLOX-5 studies. Department of Cellular and Molecular Medicine, KU C.G.-F., M.P., and B.P. performed the pharmacokinetics. M.B.- Leuven�University of Leuven, Leuven 3000, Belgium; X. and S.H.L.V. synthesized the chemical probe 28 and carried orcid.org/0000-0001-7957-2199 out off target profile experiments. S.C., E.J.C., S.O., C.M., Christian Griñán-Ferré − Pharmacology Section, Department B.D.H., F.F., and S.V. analyzed the data. S.C. wrote the first of Pharmacology, Toxicology and Therapeutic Chemistry, draft of the manuscript. S.C., C.C.-T., F.F., and S.V. wrote, Faculty of Pharmacy and Food Sciences, Institute of edited, and reviewed the manuscript with feedback from all the Neuroscience, University of Barcelona (NeuroUB), Barcelona authors. All authors have given approval to the final version of 08028, Spain; orcid.org/0000-0002-5424-9130 the manuscript. Concepción Pérez − Institute of Medicinal Chemistry, Spanish National Research Council (CSIC), Madrid 28006, Notes Spain The authors declare the following competing financial María Isabel Rodríguez-Franco − Institute of Medicinal interest(s): S.C. and S.V. are inventors of the Universitat de Chemistry, Spanish National Research Council (CSIC), Barcelona patent application on sEH inhibitors WO2019/ Madrid 28006, Spain; orcid.org/0000-0002-6500-792X 243414. C.M. and B.D.H. are inventors of the University of Antón L. Martínez − Drug Screening Platform/Biofarma California patents on sEH inhibitors licensed to EicOsis. None Research Group, CIMUS Research Center, University of of the other authors has any disclosures to declare. Santiago de Compostela (USC), Santiago de Compostela 15782, Spain; orcid.org/0000-0002-1595-3459 M. Isabel Loza − Drug Screening Platform/Biofarma Research Group, CIMUS Research Center, University of Santiago de ■ ACKNOWLEDGMENTS This work was funded by the Spanish Ministerio de Economía, Compostela (USC), Santiago de Compostela 15782, Spain; Industria y Competitividad (grants SAF2017-82771-R to S.V., orcid.org/0000-0003-4730-0863 RTI2018-093955-B-C21 to M.I.R.-F., PGC2018-102192-B-I00 Mercè Pallàs − Pharmacology Section, Department of to S.O. and RTI2018-101032-J-I00 to F.F.), the Spanish Pharmacology, Toxicology and Therapeutic Chemistry, MCIN/AEI/10.13039/501100011033 and “ERDF A way of Faculty of Pharmacy and Food Sciences, Institute of making Europe” (Grant PID2020-118127RB-I00 to S.V. and Neuroscience, University of Barcelona (NeuroUB), Barcelona Grant PID2019-106285RB to M.P. and C.S.) funded by 08028, Spain; orcid.org/0000-0003-3095-4254 MCIN/AEI/10.13039/501100011033 and by “ERDF A way Steven H. L. Verhelst − Laboratory of Chemical Biology, of making Europe”, the Xunta de Galicia (ED431G 2019/02 Department of Cellular and Molecular Medicine, KU and ED431C 2018/21), the Fundació Bosch i Gimpera, Leuven�University of Leuven, Leuven 3000, Belgium; Universitat de Barcelona (F2I grant), the Generalitat de Leibniz Institute for Analytical Sciences ISAS, AG Chemical Proteomics, Dortmund 44227, Germany; orcid.org/0000- Catalunya (2017 SGR 106 and 2017 SGR 1707), the European 0002-7400-1319 Research Council (ERC-2015-StG-679001-NetMoDEzyme to Coral Sanfeliu − Institute of Biomedical Research of Barcelona S.O.), and the European Community (MSCA-IF-2014-EF- (IIBB), CSIC and IDIBAPS, Barcelona 08036, Spain 661160-MetAccembly to F.F.). S.C. acknowledges a PhD Ferran Feixas − CompBioLab Group, Departament de fellowship from the Universitat de Barcelona (APIF grant) and Química and Institut de Química Computacional i Catàlisi the Spanish Society of Medicinal Chemistry (SEQT) and Lilly (IQCC), Universitat de Girona, Girona 17003, Spain for an “Award for Novel Researchers in the Discovery and Bruce D. Hammock − Department of Entomology and Development of New Drugs”. M.B.X. acknowledges FWO for a Nematology and Comprehensive Cancer Center, University of post-doctoral fellowship (no. 12Y0720N). S.H.L.V. acknowl- California, Davis, California 95616, United States; edges the Ministerium für Kultur und Wissenschaft des Landes orcid.org/0000-0003-1408-8317 Nordrhein-Westfalen, the Regierende Bü rgermeister von José Brea − Drug Screening Platform/Biofarma Research Berlin, and the Bundesministerium für Bildung und Forschung. Group, CIMUS Research Center, University of Santiago de F.F. acknowledges Spanish MICINN for a Ramón y Cajal Compostela (USC), Santiago de Compostela 15782, Spain; fellowship (RYC2020-029552-I). Partial support was provided orcid.org/0000-0002-5523-1979 by NIH-NIEHS River Award R35 ES03443, NIH-NIEHS Enrique J. Cobos − Department of Pharmacology, Faculty of Superfund Program P42 ES004699, NINDS R01 DK107767, Medicine and Biomedical Research Center (Neurosciences and NIDDK R01 DK103616 to B.D.H. The content is solely Institute), Biosanitary Research Institute ibs.GRANADA, the responsibility of the authors and does not necessarily University of Granada, Granada 18016, Spain represent the official views of the National Institutes of Health. Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.2c00515 Author Contributions ■ ABBREVIATIONS USED AA, arachidonic acid; aMD, accelerated molecular dynamics; S.V. conceived the idea. S.C. and B.J. synthesized and ATR, attenuated total reflectance; BFC, benzyloxytrifluorome- thylcoumarin; COX, cyclooxygenase; CYP, cytochrome P450; chemically characterized the compounds. E.J.C. designed the CTX, cyclophosphamide; DBF, dibenzylfluorescein; EETs, in vivo experiments. J.M.E. and R. G.-C. carried out the in vivo epoxyeicosatrienoic acids; EtOAc, ethyl acetate; LOX, lip- experiments. C.M. and B.D.H. performed the determination of oxygenase; MS-PPOH, N-methanesulfonyl-6-(2- the IC50 in human and murine sEH. F.F., C.C.-T., and S.O. proparyloxyphenyl)hexanamide; ND, not determined; PI, performed MD calculations. A.L.M., M.I.L., and J.M.B. carried propidium iodide; sEH, soluble epoxide hydrolase; sEHI, out DMPK studies. R.C. and C.S. performed cytotoxicity soluble epoxide hydrolase inhibitors 13678 https://doi.org/10.1021/acs.jmedchem.2c00515 J. Med. Chem. 2022, 65, 13660−13680 Journal of Medicinal Chemistry pubs.acs.org/jmc Article ■ REFERENCES (1) Harizi, H.; Corcuff, J. 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